Protection against environmental toxicity through manipulation of the processing of messenger rna precursors

ABSTRACT

This invention describes the identification of pre-messenger RNA processing as a novel target of environmental stress caused for example by lithium and sodium toxicity. Overexpression of different types of proteins (or protein fragments) from different organisms but all involved in pre-mRNA processing, protects yeast from salt stress, which indicates that any stimulation of this process, independently of its mechanism, may counteract the toxic effects of mineral salts. A similar phenotype of tolerance to NaCl and to LiCl has been observed by overexpression of these types of proteins in transgenic Arabidopsis plants, demonstrating the generality of this protective effect in eukaryotic cells and organisms.

FIELD OF THE INVENTION

[0001] This invention refers to the use of nucleic acids and proteinsinvolved in the processing of messenger RNA precursors for theenhancement of of tolerance to environmental stress such as mineral salttoxicity in eukaryotic cells and organisms.

BACKGROUND TO THE INVENTION

[0002] The nature of the cellular targets sensitive to lithium andsodium toxicity represents an important gap in our knowledge on thephysiology of ion homeostasis in eukaryoUc cells.

[0003] The characterisation of these targets is essential for theunderstanding of clinical problems such as the effects of lithium on thetherapy for dipolar disorder [Schou (1997) Arch, Gen. Psychiatry 54, 9]or high sodium levels associated with hypertension [Lifton (1996)Science 272, 676). Another problem, completely different but alsorelated to ionic homeostasis, is the progressive salinisation ofcultivated lands subjected to intensive irrigation, which has turnedcrop plant breeding for salt tolerance into an urgent need for thedevelopment of a sustainable agriculture in arid regions [Serrano (1996)Int. Rev. Cytol. 165; 1; Yeo (1998) J. Exp. Bot. 49, 915; Holmberg &Bülow (1998) Trends Plant Sci. 3, 61].

[0004] Apart from ion transport [Haro et al. (1991) FEBS Lett. 291, 189;Gaxiola et al. (1999) Proc. natl. Acad. Sci. USA 96, 1480; Apse et al.(1999) Science 285, 1256] and osmolyte synthesis [Tarczynski et al(1993) Science 259, 508; Kishor et al. (1995) Plant Physiol. 108, 1387:Alia et al. (1998) Plant J. 16, 155], the manipulation of cellularsystems most sensitive to high ion concentrations and to water stressoffers alternative routes to improve salt tolerance of crop plants[Serrano (1996) Int. Rev. Cytol. 165, 1; Tezara et al. (1999) Nature401, 914].

[0005] Genetic and biochemical analyses have allowed to identify theproduct of the yeast gene HAL2 as an important physiological target ofsalt toxicity [Gläser et al. (1993) EMBO J. 12, 3105; Dichtl et al.(1997) EMBO J. 16, 7184]. HAL2 encodes a 3′,5′-biphosphate nucleotidase,which is very sensitive to inhibition by lithium and sodium [Murguía etal. (1995) Science 267, 232]. Salt inhibition of Hal2p results in theintracellular accumulation of 3′-phosphoadenosine 5′-phosphate (pAp)[Murguía et al. (1996) J. Biol. Chem. 271, 29029], a toxic compoundwhich in turn inhibits the reactions of reduction and transfer ofsulphate groups, as well as some exoribonucleases [Dichtl et al. (1997)EMBO J. 16, 7184; Gil-Mascarell et al. (1999) Plant J. 17, 373]. Thereare genes homologous to HAL2 in plants [Gil-Mascarell et al. (1999)Plant J. 17, 373] and in mammals [López-Coronado et al. (1999) J. Biol.Chem. 274, 16043] although in the latter case the encoded enzyme isinhibited by lithium but not by sodium.

[0006] The salt tolerance conferred by overexpression of Hal2p, thewild-type protein as well as mutated versions resistant to lithium andsodium, is relatively modest [Albert et al. (2000) J. Mol. Biol. 295,927]. This suggests the existence of additional targets of salttoxicity, which become limiting once the HAL2 bottleneck is overcome,but the nature of these important salt-sensitive processes is not yetknown.

[0007] Two patent applications relating to osmotic stress but describingprotective mechanisms different to the general mechanism described inthe present invention are the following: ES2110918A (1998-02-16)relating to the production of plants tolerant to osmotic stress throughthe manipulation of carbohydrate metabolism and ES2134155A1 (1998-10-07)relating to a method to confer tolerance to osmotic, water and saltstress in glycophylic plants, through the use of genes encoding proteinswith peroxidase activity.

SUMMARY OF THE INVENTION

[0008] The technical problem underlying the present invention is toprovide a method that can be used to enhance stress tolerance of cellsand organisms that suffer from stress conditions like osmotic stress,caused by salt, drought or cold and freezing stress. A solution to thistechnical problem is achieved by providing protection against osmoticstress in cells and organisms through the manipulation of the processingof messenger RNA. Provided by the present invention is at set ofisolated genes that are able to confer to a heterologeous host cell orhost organism tolerance to stress conditions. These genes are allinvolved in the processing of mRNA precursors (such as synthesis,splicing, 5′ and 3′ end modification, movement, transport to thecytoplasm, metabolism etc. . . ) and they all showed a salt resistancephenotype when separately transformed to a salt sensitive yeast mutant.By doing so each gene acted as an efficient enhancer of stresstolerance, without the assistance of additional factors.

[0009] This set of genes, comprising SR-like proteins, (nuclear) RNAbinding factors, components of ribonucleoprotein complexes,transcription factors, and nuclear movement proteins, enables the personskilled in the art to genetically alter the organism of interest inorder to make it tolerant to stress situations such as osmotic stresssituations, more particularly mineral salt or Na+ or Li+ toxicity. Eachof the disclosed genes enables the person skilled in the art to modifycell fate and/or plant development and/or biochemistry and/or physiologyby introducing at least one of these genes into the cell. For thecultivation of crop plants for example, of which many are sensitive tostress conditions like salt, drought or cold, the disclosed genes offerthe possibility to solve the problem of reduced yield and reducedeconomic profit.

[0010] This invention offers a solution to cellular toxicity caused byenvironmental stress through the manipulation of the processing ofmessenger RNA precursors and the embodiments of the invention comprisemethods, nucleic acids, polypeptides, vectors, host cells and transgenicorganisms like transgenic plants.

DETAILED DESCRIPTION OF THE INVENTION

[0011] Soil salinity is one of the most significant abiotic orenvironmental stresses for plant agriculture. Apart from the practicalgoal of genetically improving the salt tolerance of crop plants, salttolerance research represents an important part of basic plant biology.Also research on two other major abiotic stresses, drought and cold, isintimately linked with salt stress work. For example, many genes thatare regulated by salt stress are also responsive to drought or coldstress (Zhu J. K., 1997, Molecular aspects of osmotic stress in plants,CRC Crit. Rev. Plant Sci. 16 253-277). A person skilled in the art thuscan assume that when an isolated gene confers salt tolerance to a hostorganism when transfected herein, it could also confer cold and/ordrought stress tolerance. Salt, drought and cold are considered to bethe three most important forms of osmotic stress.

[0012] In order to identify novel targets of environmental toxicity ineukaryotic cells, the inventors characterised genes conferring anincrease in the tolerance to mineral salts when expressed in yeastcells. Therefore, cDNA libraries from Arabidopsis thaliana [Minet et al.(1992) Plant J. 2, 417] and Beta vulgaris were searched which couldconfer tolerance to salt stress by overexpression in yeast cells, inwhich accumulation of pAp, and its toxic effect, was avoided by additionof methionin to the culture medium[Gläser et al. (1993) EMBO J. 12,3105; Dichtl et al. (1997) EMB J. 16, 7184; Murguía et al. (1995)Science 267, 232; Murguía et al. (1996) J. Biol. Chem. 271, 29029]. Theresult of this strategy has given rise to the present invention. This isa functional approach to identify genes and proteins that are involvedin the response of plants to salt stress. For this purpose cDNAexpression libraries were constructed as described in example 1 andexample 3 and competent yeast strains (see example 2) were used toscreen cDNAs that increased the yeast salt tolerance uponoverexpression. The growth of these yeast strains is normally inhibitedat high NaCl or LiCl concentrations of (150 mM) similar to thoseimpairing growth of most crop species. After transforming the yeastcells with the cDNA library, colonies were pooled and selected for theirability to grow in the presence of 150 mM NaC1. This screening procedureis further described in example 4.

Isolation of Arabidopsis thaliana Genes that Enhance Salt Tolerance

[0013] A cDNA library from the plant Arabidopsis thaliana of ca 7,5×10⁵transformants, was screened and three independent clones were isolatedwhich were able to confer tolerance to high salt concentrations in yeast(FIG. 1). Surprisingly, these three cDNAs encode proteins implicated inthe processing of messenger RNA precursors. These three proteins werenamed Ct-SRL1, RCY1 and U1A.

[0014] Two of these proteins belong to the family of the so-called“SR-like” or “alternating arginin-rich” factors, defined by having adomain with a high content in Arg residues alternating with Ser, Asp,andlor Glu (RS domain) (FIG. 2). Members of this family have beeninvolved in constitutive and/or alternative splicing, and in thecoupling of different steps during processing and metabolism ofmessenger RNA (transcription, modifications at the 5′ and 3′ ends, andpro-mRNA splicing, transport of mature RNA to the cytoplasm, etc.). TheCt-SRL1 protein, with amino acid composition as set forth in SEQ ID NO.3, is encoded by the cDNA identified herein as SEQ ID NO.1, which canalso be found on the genomic region of Arabidopsis thaliana chromosome5, P1 clone MNJ8 (Genbank accession number AB017069). Also the proteinsequence of SEQ ID. NO. 3 is present in the public database under theaccession number BABO9109 but no function was assigned to thissequence.The cDNA of Ct-SRL1 is not full-length and it encodes thecarboxy-terminal end of a putative SR-like protein, which includes theRS domain. Its expression in yeast confers tolerance to lithium andsodium (FIG. 1).

[0015] The second clone encodes a putative protein with an N-terminalcyclin domain related to those of K-type and T-type cyclins and with anarginin-rich domain at the C-terminus (FIG. 2A). This SR-like proteinwas named RCY1 for altering arginin-rich cyclin 1. Its expression inyeast conferred tolerance to lithium and sodium (FIG. 1) To determinethe role of the RS-domain in this SR-like protein for the enhancement ofsalt tolerance in yeast, the cyclin domain and the RS domain of RCY1were separately cloned and transformed into yeast cells. The division ofboth domains is marked in FIG. 2A by the underlined methionin residueand the C-terminal part, containing the RS domain is referred to as SEQID NO. 21 (FIG. 5). The expression of only the RS domain confers thesame phenotype as expression of the full-length protein (FIG. 3). Theseresults demonstrate that, in the case of “SR-like” factors, it is theexpression of the RS domain per se, and not of a specific protein, whichconfer salt stress tolerance. The amino acid composition of RCY1 proteinis represented herein as SEQ ID NO. 4. The cDNA of RCY1 is identifiedherein as SEQ ID NO. 2 and this sequence can be found on the genomicregion of Arabidopsis thaliana chromosome II sequence from clone T9J22.The nucleotide sequences of this genomic Arabidopsis thaliana clone,that corresponds to a part of the nucleotide sequence of the RCY1 gene,is annotated as a putative cyclin (accession number AAC14513.1). Thisprotein prediction from the public database, lacks the first 55 aminoacids of the RCY1 protein. As such, the fragment of the RCY1 proteincorresponding with the amino-terminal region is represented in SEQ ID NO22.

[0016] The third Arabidopsis clone encodes a U1A protein fromArabidopsis, a component of the U1-snRNP, the ribonucleoprotein complexwhich recognises the 5′-splice site in one of the first steps in theprocessing of introns of the pre-messenger RNA. The U1A protein ofArabidopsis is previously described by Simpson et al (1995, EMBO J. 14:4540-4550) as a specific component of the spliceosomal U1-snRNP. Theexpression of U1A in yeast conferred weaker LiCl tolerance and notolerance to NaCl (FIG. 1). The U1A protein, with amino acid compositionas set forth in SEQ ID NO 6, is encoded by the cDNA identified herein asSEQ ID. NO. 5. The protein sequence as well as the nucleic acid sequencecan be found in the public database under the accession numbersCM90283.1 and Z49991 respectively and are fully annotated. The sequencesdescribed above are presented in FIG. 5.

[0017] The phenotypes as described above were observed in the presenceand absence of methionine, and in different genetic backgrounds.

[0018] The improvement of salt tolerance by expression of theArabidopsis clones was not due to the stimulation of ion transport inyeast, since it was not associated to changes in the intracellularlithium concentrations. This was determined as described in example 5.Also the phenotypes were maintained in a yeast strain defective invacuolar transport.

[0019] The above-mentioned data suggested that an impact on anothercellular process was responsible for the observed stress tolerance. Theinventors believed that processing of messenger RNA precursors could bea target, of ionic toxicity in eukaryotic cells. This has not beendescribed previously.

[0020] In agreement herewith, the inventors have confirmed thatprocessing of introns of pre-mRNAs is inhibited in yeast in the presenceof, for example, lithium chloride. Two independent tests for pre-mRNAsplicing in vivo supported this invention. First, the inventors havemeasured the specific activity of the enzyme β-galactosidase synthesisedin yeast cells from a plasmid containing the E. coli LacZ geneartificially interrupted by an intron (example 6). They detected adecrease in the accumulation of this enzyme, as compared to thatproduced from the control construct without intron, when LiCl is addedto the culture medium. Simultaneous expression of the RS domain ofArabidopsis SRL1 in these yeast cells partially blocked the observedinhibition (data not shown). These results have been confirmed by thesecond assay, in which the inventors determined directly, by the RT-PCRtechnique as described in example 7, the inhibition of splicing in thepresence of lithium. The accumulation of endogenous yeast messenger RNAprecursors in the presence of LiCl, for example the pre-mRNAcorresponding to the SAR1 gene, was demonstrated. Because a generalinhibition of splicing would first affect the removal of those intronsnormally processed with lower efficiency, the inventors choose for theseexperiments the SAR1 pre-mRNA, which contains such an intron (Kao andSiliciano, 1996, Mol. Cell. Biol. 16: 960-967). Here again, theinventors observed the accumulation of SAR1 pre-mRNA by incubating yeastcells under salt stress conditions, and how it was partially reversed bysimultaneous co-expression of Ct-SRL1. In this way the inventorsdemonstrated that the inhibition of processing precursor mRNA in thepresence of salt is partially reverted by expression of one of theArabidopsis clones mentioned before (e.g. Ct-SRL1).

[0021] The general significance of the present invention wascorroborated by the phenotype of transgenic Arabidopsis plants, whichoverexpressed the Ct-SRL1 cDNA. Supporting the general character of themechanism, the expression of the same Ct-SRL1 cONA in under control ofthe CaMV 35S promoter Arabidopsis transgenic plants, increases theirtolerance to NACl and LiCl in a similar way as in yeast. This can beobserved, for example, by germination of transgenic seeds in agar platescontaining LiCl concentrations which are toxic to wild-type controlseeds (FIG. 4). The three independent transgenic lines were able to growindicating the efficiency of the method of the present invention.

[0022] From these results and from the very nature of the isolatedArabidopsis clones, it can be deduced that any stimulation of theprocessing of messenger RNA precursors, independently of the mechanisminvolved, counteracts the toxic effect of the salt, and that thisprotective effect against salt stress is general in all eukaryotic cellsand organisms.

[0023] The universal character of the invention, namely that theprotective effect against salt stress is not species-dependent, wasconfirmed by the isolation of sugar beet genes and eukaryotic geneswhich also confer salt tolerance and which were also related to theprocessing of messenger RNA precursors.

Isolation of Beta Vulgaris Genes that Enhance Salt Tolerance

[0024] Another aspect of the present invention is the procedure ofscreening a cDNA library from NaCl induced sugar beet leaves andsubsequent isolation of the seven sugar beet genes that confer stresstolerance to yeast cells. A functional approach to identify sugar beetgenes and proteins that are involved in the response of plants to saltstress was followed. For this purpose a NaCl-induced cDNA expressionlibrary was constructed from sugar beet leaves as described in example 1and example 3 and the Na⁺-sensitive yeast mutant strain JM26 (seeexample 2) was used to screen for sugar beet cDNAs that increased theyeast salt tolerance upon overexpression. The growth of this yeastmutant is normally inhibited at NaCl concentrations (150 mM) similar tothose impairing growth of most crop species. After transforming theyeast cells with the cDNA library, colonies were pooled and selected fortheir ability to grow in the presence of 150 mM NaCl. This screeningprocedure is further described in example 4. Six positive clones whichsurvived the high concentrations of salt, were further characterised andcontained a gene with a sequence as in SEQ ID NO.7, SEQ ID NO 9, SEQ IDNO 11, SEQ ID NO. 13, SEQ ID NO. 15 or SEQ ID NO. 17. The correspondingprotein sequences encoded by these genes have an amino acid compositionas set forth in SEQ ID NO 8, SEQ ID NO. 10, SEQ ID NO 12, SEQ ID NO. 14,SEQ ID NO. 16 or SEQ ID NO. 18 respectively. All these sequences arepresented in FIG. 5. Surprisingly, each of the six selected yeast clonesare transformed by a gene that encodes a protein which is implicated inthe processing of mRNA: one protein is a putative arginine-aspartate RNAbinding protein, two are a putative RNA binding protein, two are aputative transcription factor and one shows similarity to a nuclearmovement protein. Therefore these proteins (and the encoding genes) aresuitable to be used as stress tolerance enhancers through themanipulation of pre-mRNA processing, as is described for the Arabidopsisthaliana genes of the present invention and as described above.

[0025] The selected genes and their encoded proteins are furtherdescribed in the following paragraphs.

[0026] Accordingly, the invention relates to a novel isolated nucleicacid of red beet as set forth in SEQ ID NO. 7, encoding a putativearginine-aspartate RNA binding protein and capable of enhancing salttolerance in yeast cells. The open reading frame, starting at nucleotideposition 51 and ending at position 1079 encodes the amino acid sequenceas set forth in SEQ ID NO. 8. This polypeptide has 76% identity and 86%similarity with the Arabidopsis thaliana putative arginin-aspartate RNAbinding protein (Swiss prot accession number AAB68037). The amino acidcomparison was done with the program GAP (Symbol comparison table:blosum62. cmp, CompCheck: 6430, BLOSUM62 amino acid substitution matrix,Reference: Henikoff, S. and Henikoff, J. G. (1992). Amino acidsubstitution matrices from protein blocks. Proc. Natl. Acad. Sci. USA89: 10915-10919.). This program was also used for comparing the aminoacid sequences of the following red beet polypeptides.

[0027] The invention also relates to a novel isolated nucleic acid ofred beet as set forth in SEQ ID NO. 9, encoding a putative RNA bindingprotein and capable of enhancing salt tolerance in yeast cells. The openreading frame, starting at nucleotide position 14 and ending at position625 encodes the amino acid sequence as set forth in SEQ ID NO. 10. Thispolypeptide has 68% identity and 78% similarity with the Arabidopsisthaliana putative RNA binding protein (Swiss prot accession numberAAG52616.1).

[0028] The invention also relates to a novel isolated nucleic acid ofred beet as set forth in SEQ ID NO. 11, further referred to as clone orsequence number 10, encoding a putative transcription factor and capableof enhancing salt tolerance in yeast cells. The open reading frame,starting at nucleotide position 51 and ending at position 1119 encodesthe amino acid sequence as set forth in SEQ ID NO. 12. This polypeptidehas 81% identity and 86% similarity with the Spinacia oleracea nuclearRNA binding protein (Swiss prot accession number AAF14145.1).

[0029] Nucleotides 1 to 475 from SEQ ID NO. 11 were published in the ESTdatabase under the accession number BF011019 with the description of aDNA fragments of a Sugar beet germination cDNA library and that issimilar to a putative transcription factor.

[0030] The invention also relates to a novel isolated nucleic acid ofred beet as set forth in SEQ ID NO. 13, encoding a putativetranscription factor and capable of enhancing salt tolerance in yeastcells. The open reading frame, starting at nucleotide position 51 andending at position 1121 encodes the amino acid sequence as set forth inSEQ ID NO. 14. This polypeptide has 81% identity and 87% similarity withthe Spinacia oleracea nuclear RNA binding protein (Swiss prot accessionnumber AAF14144.1).

[0031] The invention also relates to a novel isolated nucleic acid ofred beet as set forth in SEQ ID NO. 15, encoding a putative RNA bindingprotein and capable of enhancing salt tolerance in yeast cells. The openreading frame, starting at nucleotide position 2 and ending at position970 encodes the amino acid sequence as set forth in SEQ ID NO. 16. Thispolypeptide has 68% identity and 74% similarity with the Oryza sativaputative RNA binding protein (Swiss prot accession number MG59664.1).

[0032] The invention also relates to a novel isolated nucleic acid ofred beet as set forth in SEQ ID NO. 17, encoding an unknown type ofprotein and capable of enhancing salt tolerance in yeast cells. The openreading frame, starting at nucleotide position 35 and ending at position922 encodes the amino acid sequence as set forth in SEQ ID NO. 18. Thispolypeptide has 61% identity and 69% similarity with the Arabidopsisthaliana protein with similarity to a nuclear movement protein (Swissprot accession number BAA97317.1).

[0033] Accordingly, a preferred embodiment of the present inventionrelates to a method for induction of stress tolerance to an organismcomprising the expression of a (or at least one) Beta vulgaris gene,which is involved in the processing of messenger RNA precursors.

[0034] Also the screening and selection procedure as described above canbe used to select genes from other organisms than plants. As an example,a sequence of Mus musculus was selected that enhances salt tolerance inyeast cells. This sequences is set forth in SEQ ID NO. 19 and it encodesa putative small subunit of an U2 snRNP auxiliary factor protein and iscapable of enhancing salt tolerance in yeast cells. The open readingframe, starting at nucleotide position 37 and ending at position 969encodes the amino acid sequence as set forth in SEQ ID NO. 20. Thispolypeptide has 78% identity and 84% similarity with the Arabidopsisthaliana U2 snRNP auxiliary factor, small subunit (Swiss prot accessionnumber BAB10638.1). This sequence is presented in FIG. 5.

[0035] This result illustrates that mammalian genes can also be use inthe method of the present invention. The method of the present inventionis thus generally applicable to confer stress tolerance to a host cellor organism through the manipulation of messenger RNA precursors.

[0036] The surprisingly strong phenotype of some of the yeast clonesselected as described above and the fact that these genes in an isolatedposition and in a heterologous background acted as stress toleranceenhancers, makes these genes very attractive tools to induce stresstolerance in any organism of interest, without the need for accessorycompounds. The ability of these genes to enhance osmotic stresstolerance, particularly mineral salt stress such as Na+ and Li+ stressin yeast cells when isolated and transfected herein, clearlydemonstrates their potential to confer on their own osmotic stresstolerance to any heterologeous host organism. Alternatively, each of thestress tolerance genes of the present invention can be combined withanother gene, in order to alter cell fate or plant morphology, plantdevelopment, plant biochemistry or plant physiology.

[0037] According to a first embodiment the present invention relates toa method to enhance stress tolerance in cells and organisms comprisingthe manipulation of the process of processing messenger RNA precursors.

[0038] According to a preferred embodiment, said stress could beenvironmental stress, such as but not limited to osmotic stress, saltstress, drought stress, cold or freezing stress. According to apreferred embodiment, the methods of the invention relate to theenhancement of salt tolerance of cells and organisms.

[0039] Another embodiment of the invention relates to a method toprotect cells and organisms against salt toxicity comprising themanipulation of the process of processing messenger RNA (mRNA)precursors.

[0040] According to one embodiment, one way of manipulating the processof processing messenger RNA precursors is by genetic or biochemicalmanipulation of at least one molecule which is involved in or whichinterferes with the process of processing messenger RNA precursors orwith one of the pathways of processing mRNA precursors. The term “cells”relates to any prokaryotic or eukaryotic cell. The term “organism”relates to any mono- or multicellular organism of prokaryotic oreukaryotic origin.

[0041] The present invention clearly describes several genes andproteins belonging to different classes of genes and proteins which canbe used to enhance stress tolerance. These genes and proteins of theinvention have been shown to have an effect on the process of mRNAprocessing.

[0042] Therefore, according to yet preferred embodiments, the inventionrelates to any of the above-mentioned methods wherein the genetic orbiochemical manipulation of a protein possessing a domain with a highcontent in Arg-Ser, Arg-Glu and Arg-Asp dipeptides (RS domain), an RNAbinding protein, a component of the U1-snRNP or the U2-snRNP complex, atranscription factor, or a nuclear movement protein is involved.

[0043] The invention also relates to the use of an isolated nucleic acidcomprising a nucleic acid sequence as represented in SEQ ID NO 1 with anamino acid sequence as set forth in SEQ ID NO 3, or a nucleic acid asrepresented in SEQ ID NO 2 with an amino acid sequence as set forth inSEQ ID NO 4 or SEQ ID NO 21, or a nucleic acid encoding a polypeptidecomprising the amino acid sequence represented in SEQ ID NO 22, for atleast one, or in at least one, of the above described methods. SEQ IDNOs 1 and 2 share substantial homology with genes encoding SR-likeproteins.

[0044] The invention also relates to an isolated nucleic acid comprisinga nucleic acid sequence as represented in SEQ ID NO 5 with an amino acidsequence as set forth in SEQ ID NO 6 for any of the above describedmethods. SEQ ID No 5 shares substantial homology with genes encoding acomponent of the I1-snRNP or the U2-snRP complex.

[0045] The invention also relates to an isolated nucleic acid comprisinga nucleic acid sequence as represented in any of SEQ ID NOs 7, 9, 11,13, 15, 17 or 19, with an amino acid sequence as set forth in any of SEQID NO 8, 10, 12, 14, 16, 18 or 20 for any of the above-describedmethods. SEQ ID NOs 7, 9, 13 and 15 share substantial homology withgenes encoding RNA-binding proteins. SEQ ID NO 19 shares substantialhomology with genes encoding a component of the I1-snRNP or the U2-snRPcomplex. SEQ ID NOs 11 and 17 share substantial homology with genesencoding transcriptional factors.

[0046] The invention further relates to an isolated nucleic acidencoding a protein or an immunologically active and/or functionalfragment of such a protein selected from the group consisting of:

[0047] a) a nucleic acid comprising a DNA sequence as given in any ofSEQ ID NOs 1, 2, 5, 7, 9, 11, 13, 15, 17 or 19 or the complementthereof,

[0048] b) Nucleic acid comprising the RNA sequence corresponding to anyof SEQ ID NOs 1, 2, 5, 7, 9, 11, 13, 15, 17 or 19 as in (a) or thecomplement thereof,

[0049] c) Nucleic acid specifically hybridizing tot the nucleotidesequence ad defined in (a) or (b),

[0050] d) nucleic acid encoding a polypeptide or protein with an aminoacid sequence which is at least 50%, preferably at least 60%, 70% or80%, more preferably at least 85% or 90%, most preferably 95% identicalto the polypeptide represented in any of SEQ ID NOs 3, 4, 6, 8, 10, 12,14, 16, 18, 20or 21,

[0051] e) nucleic acid encoding a polypeptide or protein comprising theamino acid sequence as given in any of SEQ ID NOs, 3, 4, 6, 8, 10, 12,14, 16, 18, 20, 21 or 22,

[0052] f) nucleic acid which is degenerated as a result of the geneticcode to a nucleotide sequence of a nucleic acid as given in any of SEQID NOs 1, 2, 5, 7, 9, 11, 13, 15, 17 or 19 or as defined (a) to (e),

[0053] g) nucleic acid which is diverging due to the differences incodon usage between the organisms to a nucleotide sequence encoding apolypeptide or protein as given in any of SEQ ID NOs 3, 4, 6, 8, 10, 12,14, 16, 18, 20 or 21 or as defined in (a) to (e),

[0054] h) nucleic acid which is diverging due to the differences inalleles encoding a polypeptide or protein as given in any of SEQ ID NOs,3, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21 or as defined in (a) to (e),

[0055] i) nucleic acid encoding an immunologically active and/orfunctional fragment of a polypeptide or protein encoded by a DNAsequence as given in any of SEQ ID NOs 1, 2, 5, 7, 9, 11, 13, 15, 17 or19 or as defined (a) to (e),

[0056] j) nucleic acid encoding a protein or polypeptide as defined inSEQ ID Nos, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21 or as defined in(a) to (i) characterized in that said sequence is DNA, cDNA, genomic DNAor synthetic DNA.

[0057] It should be understood that the present invention also relatesto any of the nucleic acids defined in a) to j) for use in any of themethods described earlier.

[0058] The invention further relates to a nucleic acid molecule of atleast 15 nucleotides in length specifically hybridizing with, orspecifically amplifying one of the nucleic acids of the invention,preferably those nucleic acids as defined in a) to j).

[0059] The invention also relates to a vector comprising a nucleic acidof the invention, wherein said vector preferably is an expression vectorwherein the nucleic acid is operably linked to one or more controlsequences allowing the expression of said sequence in prokaryotic and/oreukaryotic host cells. As such the invention also relates to a host cellcontaining a nucleic acid or a vector of the invention. Said host cellcan be chosen from a bacterial, insect, fungal, yeast, plant or animalcell.

[0060] According to yet another embodiment, the invention relates to theuse of a natural or synthetic nucleic acid encoding a protein containingan “RS domain” as defined earlier for use in any of the methods hereindescribed.

[0061] The invention also relates to the use of a natural or syntheticnucleic acid encoding a protein involved in the process of processingmessenger RNA precursors in eukaryotic cells in any of the methods ofthe invention.

[0062] The invention further relates to the use a nucleic acid with atleast 50% identity to at least one of the sequences herein described ina method for enhancing stress tolerance comprising the manipulation ofthe process or pathway of processing mRNA precursors. Preferably saidnucleic acid originates from an eukaryotic cell or organism.

[0063] Also comprised within the invention are anti-sense moleculescorresponding to at least one of the nucleic acids of the invention andtheir use in any of the methods of the invention.

[0064] According to yet another embodiment, the present inventionrelates to a polypeptide encodable by at least one of the nucleic acidsof the invention, or a homologue thereof or a derivative thereof, or animmunologically active and/or functional fragment thereof, saidpolypeptide being natural, synthetic, enriched, isolated, cell-freeand/or recombinant. Each of these polypeptides can be used in any of themethods of the invention.

[0065] Preferred polypeptides are those comprising an amino acidsequence as given in any of SEQ ID NOs 3, 4, 6, 8, 10, 12, 14, 16, 18,20, 21 or 22 a homologue thereof or a derivative thereof, or animmunologically active and/or functional fragment thereof.

[0066] The invention also relates to a method of producing a polypeptideof the invention comprising culturing a host cell as described earlierunder the conditions allowing the expression of the polypeptide andrecovering the produced polypeptide from the culture.

[0067] The invention also relates to a method for the production oftransgenic plants, plant cells or plant tissues comprising theintroduction of a nucleic acid of the invention in an expressible formator a vector of the invention in said plant, plant cell or plant tissue.

[0068] The invention also relates to a method for the production ofaltered plants, plant cells or plant tissues comprising the introductionof a polypeptide of the invention directly into a cell, a tissue or anorgan of said plant.

[0069] The invention further relates to a method for effecting theexpression of a polypeptide of the invention comprising the introductionof a nucleic acid of the invention operably linked to one or morecontrol sequences or a vector of the invention stably into the genome ofa plant cell.

[0070] The invention also relates to said methods for producingtransgenic plants, further comprising regenerating a plant from saidplant cell.

[0071] The invention further relates to a transgenic plant cellobtainable by one of the above-mentioned methods wherein said nucleicacid is stably integrated into the genome of said plant cell.

[0072] According to the invention, transgenic plants tolerant to saltstress can be produced as a result of the expression of at least one ofthe nucleic acids of claim 13 or at least one of the polypeptides ofclaim 25 or 26 or an anti-sense molecule of claim 23. Said transgenicplants are also part of the invention, as well as transgenic plantswhich as a result of the expression of at least one of the nucleic acidsor antisense molecules of the invention or at least one of thepolypeptides of the invention show an alteration of their phenotype.

[0073] The invention relates to any harvestable part of a plant of theinvention which is preferably selected from the group consisting ofseeds, leaves, fruits, stem cultures, rhizomes, roots, tubers and bulbs.Also the progeny derived from any of the plants or plant parts of theinvention are part of the present invention.

[0074] According to another embodiment the invention relates to a methodfor enhancing stress tolerance in (a) plant(s) comprising expression ofat least one of the nucleic acids or at least one of the polypeptides ananti-sense molecule of the invention in cells, tissues or parts of saidplant(s).

[0075] According to another embodiment, the invention relates to amethod for altering stress tolerance in (a) plant(s) comprisingexpression of at least one of the nucleic acids or at least one of thepolypeptides or an ant-sense molecule of the invention in cells, tissuesor parts of said plant(s).

[0076] It should be clear that the stress tolerance in the above methodscan mean any stress caused by the environment such as, but not limitedto osmotic stress, salt stress, drought stress, freezing stress or coldstress.

[0077] Furthermore, any of the methods, the nucleic acids, or antisensemolecules or the polypeptides of the invention can be used (in a method)for increasing yield, for stimulating growth which can be in any part ofthat plant, such as root, leave, seed.

[0078] The invention also relates to a plant obtainable by any of theabove described methods for culturing on soil with high saltconcentrations, preferably soils with a salt content of more than 1 mMsalt ions.

[0079] Also forming part of the invention are new strains of yeast orother unicellular eukaryotes more tolerant to salt stress as a result ofthe expression of any of the nucleic acids and/or proteins of theinvention.

[0080] The invention further relates to an in vitro cell culture systemcomprising animal, plant or host cells as defined earlier, tolerant tosalt, obtained as a result of the expression of at least one of thenucleic acids, vectors, polypeptides, or antisense molecule of claim 23.

[0081] The invention further relates to at least one therapeuticapplication in humans derived from the methods described herein.

[0082] The invention also relates to an antibody specificallyrecognizing a polypeptide of the invention or a specific epitopethereof.

[0083] The invention further relates to a diagnostic compositioncomprising at least a nucleic acid a vector, an antisense molecule, apolypeptide or an antibody of the invention.

Definitions and Elaboration to the Embodiments Specific Definitions

[0084] “Manipulation of a process” herein means the interference with orthe modulation of that process, preferably enhancing, catalysing,changing or altering that process. This interference can have an impacton every step or every component or every product or every result ofthat process. Also this interference can have an impact on theefficiency, the rate or the yield of that process.

[0085] “Genetic manipulation of a process” herein refers to themanipulation of a process by any kind of interfering with the geneticsequences (e.g. nucleotide sequences, RNA, DNA) that are involved inthat process. Next to the natural genetic activity of the cell such asreplication, transcription translation, and the processing of differentnucleic acids, also molecular biology techniques and gentechnologytechniques comprised in the term “genetic manipulation of a process”.These artificial genetic techniques are know by the person skilled inthe art and are for example cloning, transforming, recombining,expressing, overexpressing, silencing etc. As an example of “geneticmanipulation of a process” one can interfere with the genetic sequenceencoding a protein, which is involved in the process of processingmessenger RNA precursors. Altematively, one can interfere with an RNAmolecule (such as a small nucleolar RNA), which is directly involved inthe process of processing messenger RNA precursors.

[0086] In the case that said genetic sequence encodes a protein and theexpression, the constitution, the structure or the location of thatcoding sequence is altered, the term “genetic manipulation of a protein”is used. More particularly one can alter the composition or theexpression of said coding sequences or one can introduce or delete saidcoding sequence in the host cell, which performs said process. Morespecifically said protein can be any component which is involved in theprocessing of pre-mRNA, such as but not limited to a component of theU1-snRNP or U2-snRNP complex, a transcription factor, an RNA bindingprotein or a nuclear movement protein.

[0087] “Biochemical manipulation of a process” herein refers to themanipulation of said process by using biochemical methods that interferewith said process. With biochemical methods is meant the use of anysubstance (chemicals, peptides, and molecules) which have an impact onbiological processes. For example one can introduce a peptide, or aprotein, or a biochemical or a chemical substance in the cell performingthat process, in order to interfere with that process. More particularlyone can introduce a protein or a peptide derived from the genes of thepresent invention directly into the cell, in order to enhance theprocess of processing messenger RNA precursors.

[0088] In the case that said biochemical method involves the use of aprotein or peptide or in case said biochemical method results in themodification of a protein, we use the term “biochemical manipulation ofa protein”.

[0089] “Cells” herein is to be taken in its broadest context andincludes every living cell, such as prokaryotic and eukaryotic cells.

[0090] “Messenger RNA precursor” herein refers to any RNA molecule,which is not yet operational as a mature messenger RNA, from whichpolypeptides can be transcribed, because its composition, its structureor its location has to be changed. “Processing of messenger RNAprecursors” herein refers to any process, which changes the composition,the structure or the location of a messenger RNA precursor. Examples ofsuch processes in an eukaryotic cell are the synthesis of the messengerRNA precursor during the transcription, the modification of the 5′and/or the 3′ ends of the precursor like polyadenylation, the splicingof introns from the precursor like the constitutive or the alternativesplicing, the metabolism of precursor or the translocation of theprecursor towards the nuclear envelop and the transport of the matureRNA to the cytoplams etc. Also the coupling of the different steps ofthis processing or metabolism of the precursor DNA are part of the term“processing of messenger RNA precursors” as used in this description.Post-transcriptional processing of precursor RNA comprises a complexpathway of endonucleolytic cleavages, exonucleolytic digestion andcovalent modifications. The general order of the various processingsteps is well conserved in eukaryotic cells, but the underlyingmechanisms are largely unknown. The pre-mRNA processing is an importantcellular activity and has been studied in the yeast cells Saccharomycescerevisiae by Venema and Tollervey (Yeast, (1995) 11(16): 1629-1650).The processing steps involve a variety of protein-protein interactionsas well as protein-RNA and RNA-RNA interactions. The precise role ofdifferent transcription factors, RNA binding proteins, and other nuclearproteins such as nuclear movement proteins and nuclear RNA's in theprocessing of pre-mRNA remains largely unknown. Therefor a proteininterfering with the process of processing messenger RNA precursors, canbe a protein of many different kinds. The nuclear transport of pre-mRNAinvolved several factors which can be used in the method of the presentinvention, since they are involved in the processing of pre-mRNA.Pre-mRNA is transcribed primarily from genes located at the interfacebetween chromatin domains and the interchromatin space. After partial orcomplete processing and complexing with nuclear proteins, thetranscripts leave their site of synthesis and travel through theinterchromatin space to the nuclear pores, where they are captured bythe export machinery for export to the cytoplasm. Transport-competentmRNA's are complexed with the correct complement of nuclear proteins(reviewed in Politz and Pederson, J. struct. Biol 2000, 129(2-3):252-257). The role of the U1-snRNP and the U2-snRNP is mainly situatedin the splicing of pre-mRNA. The product of the U1 small nuclearribonucleoprotein particle or complex U1-snRNP 70K (U1-70K) gene, a U1sn-RNP-specific protein, has been implicated in basic as well asalternative splicing or pre-mRNA in animals as well as in plants(Golovkin and Reddy, Plant Cell, 1996, (8): 1421-1435). In other reportsdifferent interacting proteins of the U1-70K protein are described, suchas for example SC35-like protein and serine/arginine-rich protein(Golovkin and Reddy, J. Biol Chem 1999, 245(51): 36428-36438). For theU2 small nuclear ribonucleoprotein auxiliary factors such as U2A havebeen described (Domon et al, J. Biol Chem 1998: 273(51): 34603-34610).Within the scope of the present invention are any proteins, which areinvolved in splicing of the messenger RNA-precursors. The role of smallnucleolar RNA's in the processing of pre-mRNA in plants has beendescribed (Brown and Shaw, the Plant Cell, 1998, 10: 649-657). Thereformanipulation of the process of processing pre-mRNA can also beestablished by interfering with RNA molecules. Jarrous et al (J. CellBiol. 1999, 146(3): 559-572) showed that Rpp29 and Rpp38 (proteinsubunits of human RNaseP) are found in the nucleolus and that theyreside in coiled bodies, organelles that are implicated in thebiogenesis of several other small nuclear ribonucleoproteins requiredfor the processing of precursor mRNA. These kinds of proteins which arepart of a Ribonucleoprotein Ribonuclease complex are involved in theprocessing of pre-mRNA can therefor also be used in the method of thepresent invention. The role of nuclear movement proteins is not wellestablished in the art. Still a lot of molecules involved in theprocessing of messenger RNA precursors remain to be elucidated.

[0091] “SR-like proteins” as used herein are described previously in inBlencowe et al. 1999, 77(4): 277-291: “The processing of messenger RNAprecursors (pre-mRNA) to mRNA requires a large number of proteins thatcontains domains rich in alternating arginine and serine residues (RSdomains). These include members of the SR family of splicing factors andproteins that are structurally and functionally distinct from the SRfamily collectively referred to below as SR-related proteins. Bothgroups of RS domain proteins function in constitutive and regulatedpre-mRNA splicing. Recently, several SR-related proteins have beenidentified that are associated with the transcriptional machinery. OtherSR-related proteins are associated with mRNA 3′ end formation and havebeen implicated in export”. The evidence that proteins containing RSdomains may play a fundamental role in the co-ordination of differentsteps in the synthesis and processing of pre-mRNA is further reviewed inBlencowe et al. 1999, 77(4): 277-291.

General Definitions

[0092] The terms “protein(s)”, “peptide(s)” or “oligopeptide(s)” orpolypeptide, when used herein refer to amino acids in a polymeric formof any length. Said terms also include known amino acid modificationssuch as disulphide bond formation, cysteinylation, oxidation,glutathionylation, methylation, acetylation, farnesylation,biotinylation, stearoylation, formylation, lipoic acid addition,phosphorylation, sulphation, ubiquitination, myristoylation,palmitoylation, geranylgeranylation, cyclization (e.g. pyroglutamic acidformation), oxidation, deamidation, dehydration, glycosylation (e.g.pentoses, hexosamines, N-acetylhexosamines, deoxyhexoses, hexoses,sialic acid etc.), acylation and radiolabels (e.g. ¹²⁵I, ¹³¹I, ³⁵S, ¹⁴C,³²P, ³³P, ³H) as well as non-naturally occurring amino acid residues,L-amino acid residues and D-amino acid residues.

[0093] “Homologues” or “Homologs of a protein of the invention are thosepeptides, oligopeptides, polypeptides, proteins and enzymes whichcontain amino acid substitutions, deletions andlor additions relative tothe said protein with respect to which they are a homologue withoutaltering one or more of its functional properties, in particular withoutreducing the activity of the resulting. For example, a homologue of saidprotein will consist of a bioactive amino acid sequence variant of saidprotein. To produce such homologues, amino acids present in the saidprotein can be replaced by other amino acids having similar properties,for example hydrophobicity, hydrophilicity, hydrophobic moment,antigenicity, propensity to form or break a-helical structures orβ-sheet structures, and so on.

[0094] Substitutional variants of a protein of the invention are thosein which at least one residue in said protein amino acid sequence hasbeen removed and a different residue inserted in its place. Amino acidsubstitutions are typically of single residues, but may be clustereddepending upon functional constraints placed upon the polypeptide;insertions will usually be of the order of about 1-10 amino acidresidues and deletions will range from about 1-20 residues. Preferably,amino acid substitutions will comprise conservative amino acidsubstitutions, such as those described supra.

[0095] Insertional amino acid sequence variants of a protein of theinvention are those in which one or more amino acid residues areintroduced into a predetermined site in said protein. Insertions cancomprise amino-terminal and/or carboxy-terminal fusions as well asintra-sequence insertions of single or multiple amino acids. Generally,insertions within the amino acid sequence will be smaller than amino orcarboxyl terminal fusions, of the order of about 1 to 10 residues.Examples of amino- or carboxy-terminal fusion proteins or peptidesinclude the binding domain or activation domain of a transcriptionalactivator as used in the yeast two-hybrid system, phage coat proteins,(histidine)₆-tag, glutathione S-transferase, protein A, maltose-bindingprotein, dihydrofolate reductase, Tag•100 epitope (EETARFQPGYRS), c-mycepitope (EQKLISEEDL), FLAG®-epitope (DYKDDDK), lacZ, CMP(calmodulin-binding peptide), HA epitope (YPYDVPDYA), protein C epitope(EDQVDPRLIDGK) and VSV epitope (YTDIEMNRLGK).

[0096] Deletional variants of a protein of the invention arecharacterised by the removal of one or more amino acids from the aminoacid sequence of said protein.

[0097] Amino acid variants of a protein of the invention may readily bemade using peptide synthetic techniques well known in the art, such assolid phase peptide synthesis and the like, or by recombinant DNAmanipulations. The manipulation of DNA sequences to produce variantproteins, which manifest as substitutional, insertional or deletionalvariants are well known in the art. For example, techniques for makingsubstitution mutations at predetermined sites in DNA having knownsequence are well known to those skilled in the art, such as by M13mutagenesis, T7-Gen in vitro mutagenesis kit (USB, Cleveland, Ohio),QuickChange Site Directed mutagenesis kit (Stratagene, San Diego,Calif.), PCR-mediated site-directed mutagenesis or other site-directedmutagenesis protocols. Another alternative to manipulate DNA sequencesto produce variant proteins, which manifest as substitutional,insertional or deletional variants comprises targeted in vivo genemodification which can be achieved by chimeric RNA/DNA oligonucleotidesas described by e.g. (Palmgren 1997;Yoon et al. 1996).

[0098] “Derivatives” of a protein of the invention are those peptides,oligopeptides, polypeptides, proteins and enzymes which comprise atleast about five contiguous amino acid residues of said polypeptide butwhich retain the biological activity of said protein. A “derivative” mayfurther comprise additional naturally-occurring, altered glycosylated,acylated or non-naturally occurring amino acid residues compared to theamino acid sequence of a naturally-occurring form of said polypeptide.Alternatively or in addition, a derivative may comprise one or morenon-amino acid substituents compared to the amino acid sequence of anaturally-occurring form of said polypeptide, for example a reportermolecule or other ligand, covalently or non-covalently bound to theamino acid sequence such as, for example, a reporter molecule which isbound thereto to facilitate its detection.

[0099] With “immunologically active” is meant that a molecule orspecific fragments thereof such as epitopes or haptens are recognisedby, i.e. bind to antibodies.

[0100] In the context of the current invention are also includedhomologous, derivatives and/or immunologically active fragments of anyof the inventive polypeptides. “Antibodies” include monoclonal,polyclonal, synthetic or heavy chain camel antibodies as well asfragments of antibodies such as Fab, Fv or scFv fragments. Monoclonalantibodies can be prepared by the techniques as described previouslye.g. (Liddle & Cryer 1991) which comprise the fusion of mouse myelomacells to spleen cells derived from immunised animals. The term“antibodies” furthermore includes derivatives thereof such as labelledantibodies. Antibody labels include alkaline phosphatase, PKH2, PKH26,PKH67, fluorescein (FITC), Hoechst 33258, R-phycoerythrin (PE),rhodamine (TRITC), Quantum Red, Texas Red, Cy3, biotin, agarose,peroxidase, gold spheres and radiolabels (e.g. ¹²⁵I, ¹³¹I, ³⁵S, ¹⁴C,³²P, ³³P, ³H). Tools in molecular biology relying on antibodies againsta protein include protein gel blot analysis, screening of expressionlibraries allowing gene identification, protein quantitative methodsincluding ELISA and RIA, immunoaffinity purification of proteins,immunoprecipitation of proteins e.g. (Magyar et al 1997) andimmunolocalization. Other uses of antibodies and especially of peptideantibodies include the study of proteolytic processing (Loffler et al1994; Woulfe et al. 1994), determination of protein active sites (Lerner1982), the study of precursor and post-translational processing (Baron &Baltimore 1982;Lerner et al 1981;Semler et al. 1982), identification ofprotein domains involved in protein-protein interactions (Murakami etal. 1992) and the study of exon usage in gene expression (Tamura et al.1991).

[0101] In the scope of the current invention are also antibodiesrecognising the proteins of the present invention or homologue,derivative or fragment thereof as defined supra.

[0102] The terms “gene(s)”, “polynucleotide(s)”, “nucleic acid,sequence(s)”, “nucleotide sequence(s)”, “DNA sequence(s)” or “nucleicacid molecule(s)”, when used herein refer to nucleotides, eitherribonucleotides or deoxytibonucleobdes or a combination of both, in apolymeric form of any length. Said terms furthermore includedouble-stranded and single-stranded DNA and RNA. Said terms also includeknown nucleotide modifications such as methylation, cyclization and‘caps’ and substitution of one or more of the naturally occurringnucleotides with an analogue such as inosine. Said terms also encompasspeptide nucleic acids (PNAs), a DNA analogue in which the backbone is apseudopeptid e consisting of N-(2-aminoethyl)-glycine units rather thana sugar. PNAs mimic the behaviour of DNA and bind complementary nucleicacid strands. The neutral backbone of PNA results in stronger bindingand greater specificity than normally achieved. In addition, the uniquechemical, physical and biological properties of PNA have been exploitedto produce powerful biomolecular tools, anti-sense and anti-gene agents,molecular probes and biosensors.

[0103] With “recombinant DNA molecule” or “chimeric gene” is meant ahybrid DNA produced by joining pieces of DNA from different sources.With “heterologous nucleotide sequence” is intended a sequence that isnot naturally occurring with the promoter sequence. While thisnucleotide sequenceris heterologous to the promoter sequence, it may behomologous, or native, or heterologous, or foreign, to the plant host.“Sense strand” refers to the strand of a double-stranded DNA moleculethat is homologous to a mRNA transcript thereof. The “anti-sense strand”contains an inverted sequence, which is complementary to that of the“sense strand”.

[0104] A “coding sequence” or “open reading frame” or “ORF” is definedas a nucleotide sequence that can be transcribed into mRNA and/ortranslated into a polypeptide when placed under the control ofappropriate regulatory sequences, i.e. when said coding sequence or ORFis present in an expressible format. Said coding sequence of ORF isbounded by a 5′ translation start codon and a 3′ translation stop codon.A coding sequence or ORF can include, but is not limited to RNA, mRNA,cDNA, recombinant nucleotide sequences, synthetically manufacturednucleotide sequences or genomic DNA. Said coding sequence or ORF can beinterrupted by intervening nucleic acid sequences. Genes and codingsequences essentially encoding the same protein but isolated fromdifferent sources can consist of substantially divergent nucleic acidsequences. Reciprocally, substantially divergent nucleic acid sequencescan be designed to effect expression of essentially the same protein.Said nucleic acid sequences are the result of e.g. the existence ofdifferent alleles of a given gene, or of the degeneracy of the geneticcode or of differences in codon usage. Thus amino acids such asmethionine and tryptophan are encoded by a single codon whereas otheramino acids such as arginine, leucine and serine can each be translatedfrom up to six different codons. Differences in preferred codon usageare illustrated below for Agrobacterium tumefaciens (a bacterium), A.thaliana, M. sativa (two dicotyledonous plants) and Oryza sativa (amonocotyledonous plant). These examples were extracted from(http://www.kazusa.orjp/codon). To give one example, the codon GGC (forglycine) is the most frequently used codon in A. tumefaciens (36.2%), isthe second most frequently used codon in O. sativa but is used at muchlower frequencies in A. thaliana and M. sativa (9% and 8.4%,respectively). Of the four possible codons encoding glycine (see Table2), said GGC codon is most preferably used in A. tumefaciens and O.sativa. However, in A. thaliana this is the GGA (and GGU) codon whereasin M. sativa this is the GGU (and GGA) codon. Allelic variants arefurther defined as to comprise single nucleotide polymorphisms (SNPs) aswell as small insertionideletion polymorphisms (INDELs; the size ofINDELs is usually less than 100. bp). SNPs and INDELs form the largestset of sequence variants in naturally occurring polymorphic strains ofmost organisms. They are helpful in mapping genes and discovery of genesand gene functions. They are furthermore helpful in identification ofgenetic loci, e.g. plant genes, involved in determining processes suchas growth rate, plant size and plant yield, plant vigor, diseaseresistance, stress tolerance etc. Many techniques are nowadays availableto identify SNPs and/or INDELs including (i) PCR followed by denaturinghigh performance liquid chromatography (DHPLC; e.g. . (Cho et al.1999)); (ii) constant denaturant capillary electrophoresis (CDCE)combined with high-fidelity PCR (e.g. (U-Sucholeiki et al. 1999)); (iii)denaturing gradient gel electrophoresis (e.g. Fischer and Lerman 1983);(iv) matrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF MS; e.g. (Ross et al. 2000)); (v) real-timefluorescence monitoring PCR assays (e.g. Tapp et al. 2000); (vi)Acrydite™ gel technology (e.g. Kenney et al. 1998); (vii) cycle dideoxyfingerprinting (CddF; e.g. (Langemeier et al. 1994); (viii)single-strand conformation polymorphism (SSCP) analysis (e.g.(Vidal-Puig & Moller 1994)) and (ix) mini-sequencing primer extensionreaction (e.g. Syvanen 1999). The technique of ‘Targeting Induced LocalLesions in Genomes’ (TILLING: (McCallum et al. 2000a;McCallum et al.2000b)), which Is a variant of (i) supra, can also be applied to rapidlyidentify an altered gene in e.g. chemically mutagenized plantindividuals showing interesting phenotypes

[0105] “Hybridisation” is the process wherein substantially homologouscomplementary nucleotide sequences anneal to each other. Thehybridisation process can occur entirely in solution, i.e. bothcomplementary nucleic acids are in solution. Tools in molecular biologyrelying on such a process include the polymerase chain reaction (PCR;and all methods based thereon), subtractive hybridisation, random primerextension, nuclease S1 mapping, primer extension, reverse transcription,cDNA synthesis, differential display of RNAs, and DNA sequencedetermination. The hybridisation process can also occur with one of thecomplementary nucleic acids immobilised to a matrix such as magneticbeads, Sepharose beads or any other resin. Tools in molecular biologyrelying on such a process include the isolation of poly (A+) mRNA. Thehybridisation process can furthermore occur with one of thecomplementary nucleic acids immobilised to a solid support such as anitro-cellulose or nylon membrane or immobilised by e.g.photolithography to e.g. a siliceous glass support (the latter known asnucleic acid arrays or microarrays or as nucleic acid chips). Tools inmolecular biology relying on such a process include RNA and DNA gel blotanalysis, colony hybridisation, plaque hybridisation, in situhybridisation and microarray hybridisation. In order to allowhybridisation to occur, the nucleic acid molecules are generallythermally or chemically denatured to melt a double strand into twosingle strands and/or to remove hairpins or other secondary structuresfrom single stranded nucleic acids. The stringency of hybridisation isinfluenced by conditions such as temperature, salt concentration andhybridisation buffer composition. High stringency conditions forhybridisation include high temperature and/or low salt concentration(salts include NaCl and Na₃-citrate) andlor the inclusion of formamidein the hybridisation buffer and/or lowering the concentration ofcompounds such as SDS (detergent) in the hybridisation buffer and/orexclusion of compounds such as dextran sulphate or polyethylene glycol(promoting molecular crowding) from the hybridisation buffer.Conventional hybridisation conditions are described e.g. (Sambrook etal. 1989) but the skilled craftsman will appreciate that numerousdifferent hybridisation conditions can be designed in function of theknown or the expected homology and/or length of the nucleic acidsequence. With specifically hybridising is meant hybridising understringent conditions. Sufficiently low stringency hybridisationconditions are particularly preferred to isolate nucleic acidsheterologous to the DNA sequences of the invention defined supra.Elements contributing to said heterology include allelism, degenerationof the genetic code and differences in preferred codon usage asdiscussed supra.

[0106] Accordingy, the scope of the current invention is also related tothe use of the inventive DNA sequences encoding the polypeptides of thepresent invention, homologue, derivative and/or immunologically fragmentthereof as defined higher in any method of hybridisation. The currentinvention furthermore also relates to DNA sequences hybridising to saidinventive DNA sequences.

[0107] “Specifically amplifying” herein using an amplification methodwhich is selective and only amplifies a nucleic acid sequence with aspecific base-pair composition (e.g. polymerase chain reaction).

[0108] DNA sequences as defined in the current invention can beinterrupted by intervening sequences. With “intervening sequences” ismeant any nucleic acid sequence which disrupts a coding sequencecomprising said inventive DNA sequence or which disrupts the expressibleformat of a DNA sequence comprising said inventive DNA sequence. Removalof the intervening sequence restores said coding sequence or saidexpressible format. Examples of intervening sequences include introns,mobilizable DNA sequences such as transposons and DNA tags such as e.g.a T-DNA. With “mobilizable DNA sequence” is meant any DNA sequence thatcan be mobilised as the result of a recombination event.

[0109] To effect expression of a protein in a cell, tissue or organ,preferably of plant origin, either the protein may be introduceddirectly to said cell, such as by microinjection or ballistic means orafternatively, an isolated hucleic acid molecule encoding said proteinmay be introduced into said cell, tissue or organ in an expressibleformat.

[0110] Preferably, the DNA sequence of the invention comprises a codingsequence or open reading frame (ORF) encoding a protein of the presentinvention or a homologue or derivative thereof or an immunologicallyactive thereof as defined supra. The preferred proteins of the inventioncomprises the amino acid sequence as presented in SEQ ID NO. 3, 4, 6, 8,10, 12, 14, 16, 18, 20 and 21.

[0111] With “vector” or “vector sequence” is meant a DNA sequence, whichcan be introduced in an organism by transformation and can be stablymaintained in said organism. Vector maintenance is possible in e.g.cultures of Escherichia coli, A. tumefaciens, Saccharomyces cerevisiaeor Schizosaccharomyces pombe. Other vectors such as phagemids and cosmidvectors can be maintained and multiplied in bacteria and/or viruses.Vector sequences generally comprise a set of unique sites recognised byrestriction enzymes, the multiple cloning site (MCS), wherein one ormore non-vector sequence(s) can be inserted.

[0112] With “non-vector sequence” is accordingly meant a DNA sequencewhich is integrated in one or more of the sites of the MCS comprisedwithin a vector.

[0113] “Expression vectors” form a subset of vectors which, by virtue ofcomprising the appropriate regulatory sequences enabling the creation ofan expressible format for the inserted non-vector sequence(s), thusallowing expression of the protein encoded by said non-vectorsequence(s). Expression vectors are known in the art enabling proteinexpression in organisms including bacteria (e.g. E. coli), fungi (e.g.S. cerevisiae, S. pombe, Pichia pastoris), insect cells (e.g.baculoviral expression vectors), animal cells (e.g. COS or CHO cells)and plant cells (e.g. potato virus X-based expression vectors, see e.g.Vance et al. 1998—W09844097). See also further in this specification fortypical plant expression vectors.

[0114] The current invention clearly includes any vector or expressionvector comprising a non-vector DNA sequence comprising the nucleotidesequences according to the present invention or a non-vector sequenceencoding the proteins of the present invention, or the homologue,derivative and/or immunologically active fragment thereof as definedsupra.

[0115] As an alternative to expression vector-mediated proteinproduction in biological systems, chemical protein synthesis can beapplied. Synthetic peptides can be manufactured in solution phase or insolid phase. Solid phase peptide synthesis (Merrifield 1963) is,however, the most common way and involves the sequential addition ofamino acids to create a linear peptide chain.

[0116] By “expressible format” or “under the control of expressioncontrol sequences” is meant that the isolated nucleic acid molecule isin a form suitable for being transcribed into mRNA and/or translated toproduce a protein, either constitutively or following induction by anintracellular or extracellular signal, such as an environmental stimulusor stress (mitogens, anoxia, hypoxia, temperature, salt, light,dehydration, etc) or a chemical compound such as IPTG(isopropyl-β-D-thiogalactopyranoside) or such as an antibiotic(tetracycline, ampicillin, rifampicin, kanamycin), hormone (e.g.gibberellin, auxin, cytokinin, glucocorticoid, brassinosteroid,ethylene, abscisic acid etc), hormone analogue (iodoacetic acid (IAA),2,4-D, etc), metal (zinc, copper, iron, etc), or dexamethasone, amongstothers. As will be known to those skilled in the art, expression of afunctional protein may also require one or more post-translationalmodifications, such as glycosylation, phosphorylation,dephosphorylation, or one or more protein-protein interactions, amongstothers. All such processes are included within the scope of the term“expressible format”.

[0117] Preferably, expression of a protein in a specific cell, tissue,or organ, preferably of plant origin, is effected by introducing andexpressing an isolated nucleic acid molecule encoding said protein, suchas a cDNA molecule, genomic gene, synthetic oligonucleotide molecule,mRNA molecule or open reading frame, to said cell, tissue or organ,wherein said nucleic acid molecule is placed operably in connection withsuitable regulatory sequences including a promoter, preferably aplant-expressible promoter, and a terminator sequence.

[0118] “Regulatory sequence” refers to control DNA sequences, which arenecessary to affect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism. In prokaryotes, control sequences generally includepromoters, ribosomal binding sites, and terminators. In eukaryotesgenerally control sequences include promoters, terminators and enhancersor silencers.

[0119] Within the scope of the invention are also the nucleotidesequences as defined in the present invention fused to any regulatoryseqeuence. The term “control sequences” is intended to include, at aminimum, all components the presence of which are necessary forexpression, and may also include additional advantageous components andwhich determines when, how much and where a specific gene is expressed.

[0120] Reference herein to a “promoter” is to be taken in its broadestcontext and includes the transcriptional regulatory sequences derivedfrom a classical eukaryotic genomic gene, including the TATA box whichis required for accurate transcription initiation, with or without aCCMT box sequence and additional regulatory elements (i.e. upstreamactivating sequences, enhancers and silencers) which alter geneexpression in response to developmental and/or external stimuli, or in atissue-specific manner.

[0121] The term “Promoter” also includes the transcriptional regulatorysequences of a classical prokaryotic gene, in which case it may includea −35 box sequence and/or a −10 box transcriptional regulatorysequences.

[0122] The term “promoter” is also used to describe a synthetic orfusion molecule or derivative, which confers, activates or enhancesexpression of a nucleic acid molecule in a cell, tissue or organ.

[0123] Promoters may contain additional copies of one or more specificregulatory elements, to further enhance expression and/or to alter thespatial expression and/or temporal expression of a nucleic acid moleculeto which it is operably connected. Such regulatory elements may beplaced adjacent to a heterologous promoter sequence to drive expressionof a nucleic acid molecule in response to e.g. copper, glucocorticoids,dexamethasone, tetracycline, gibberellin, cAMP, abscisic acid, auxin,wounding, ethylene, jasmonate or salicylic acid or to confer expressionof a nucleic acid molecule to specific cells, tissues or organs such asmeristems, leaves, roots, embryo, flowers, seeds or fruits. In thecontext of the present invention, the promoter preferably is aplant-expressible promoter sequence. Promoters, however, that alsofunction or solely function in non-plant cells such as bacteria, yeastcells, insect cells and animal cells are not excluded from theinvention. By “plant-expressible” is meant that the promoter sequence,including any additional regulatory elements added thereto or containedtherein, is at least capable of inducing, conferring, activating orenhancing expression in a plant cell, tissue or organ, preferably amonocotyledonous or dicotyledonous plant cell, tissue, or organ. Theterms “plant-operable” and “operable in a plant” when used herein, inrespect of a promoter sequence, shall be taken to be equivalent to aplant-expressible promoter sequence.

[0124] In the present context, a “regulated promoter” or “regulatablepromoter sequence” is a promoter that is capable of conferringexpression on a structural gene in a particular cell, tissue, or organor group of cells, tissues or organs of a plant, optionally underspecific conditions, however does generally not confer expressionthroughout the plant under all conditions. Accordingly, a regulatablepromoter sequence may be a promoter sequence that confers expression ona gene to which it is operably connected in a particular location withinthe plant or alternatively, throughout the plant under a specific set ofconditions, such as following induction of gene expression by a chemicalcompound or other elicitor. Preferably, the regulatable promoter used inthe performance of the present invention confers expression in aspecific location within the plant, either constitutively or followinginduction, however not in the whole plant under any circumstances.Included within the scope of such promoters are cell-specific promotersequences, tissue-specific promoter sequences, organ-specific promotersequences, cell cycle specific gene promoter sequences, induciblepromoter sequences and constitutive promoter sequences that have beenmodified to confer expression in a particular part of the plant at anyone time, such as by integration of said constitutive promoter within atransposable genetic element (Ac, Ds, Spm, En, or other transposon).Those skilled in the art will be aware that an “inducible promoter” is apromoter the transcriptional activity of which is increased or inducedin response to a developmental, chemical, environmental, or physicalstimulus. Within the scope of the present invention are the nucleotidesequences as defined in the claims, fused to a stress induciblepromoter. Similarly, the skilled craftsman will understand that a“constitutive promoter” is a promoter that is transcriptionally activethroughout most, but not necessarily all parts of an organism,preferably a plant, during most, but not necessarily all phases of itsgrowth and development. Contrarily the term “ubiquitous promoter” istaken to indicate a promoter that is transcriptionally active throughoutmost, but not necessarily all parts of an organism, preferably a plant.

[0125] Those skilled in the art will readily be capable of selectingappropriate promoter sequences for use in regulating appropriateexpression of the proteins of the present inventio as described suprafrom publicly-available or readily-available sources, without undueexperimentation.

[0126] Placing a nucleic acid molecule under the regulatory control of apromoter sequence, or in operable connection with a promoter sequencemeans positioning said nucleic acid molecule such that expression iscontrolled by the promoter sequence. A promoter is usually, but notnecessarily, positioned upstream, or at the 5′-end, and within 2 kb ofthe start site of transcription, of the nucleic acid molecule which itregulates. In the construction of heterologous promoter/structural genecombinations it is generally preferred to position the promoter at adistance from the gene transcription start site that is approximatelythe same as the distance between that promoter and the gene it controlsin its natural setting (i.e., the gene from which the promoter isderived).

[0127] “Expression” means the production of a protein or nucleotidesequence in the cell itself or in a cell-free system. It includestranscription into an RNA product, post-transcriptional modificationand/or translation to a protein product or polypeptide from a DNAencoding that product, as well as possible post-translationalmodifications.

[0128] “Operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences. In case the control sequence is a promoter, it is obvious fora skilled person that double-stranded nucleic acid is preferably used.

[0129] The term “terminator” refers to a DNA sequence at the end of atranscriptional unit which signal termination of transcription.Terminators are 3′-non-translated DNA sequences containing apolyadenylation signal, which facilitates the addition of polyadenylatesequences to the 3′-end of a primary transcript. Terminators active incells derived from viruses, yeasts, moulds, bacteria, insects, birds,mammals and plants are known and described in the literature. They maybe isolated from bacteria, fungi, viruses, animals and/or plants.

[0130] Examples of terminators particularly suitable for use in the geneconstructs of the present invention include the Agrobacteriumtumefaciens nopaline synthase (NOS) gene terminator, the Agrobacteriumtumefaciens octopine synthase (OCS) gene terminator sequence, theCauliflower mosaic virus (CaMV) 35S gene terminator sequence, the Oryzasativa ADP-glucose pyrophosphorylase terminator sequence (t3′Bt2), theZea mays zein gene terminator sequence, the rbcs-1A gene terminator, andthe rbcs-3A gene terminator sequences, amongst others.

[0131] Those skilled in the art will be aware of suitable promotersequences and terminator sequences which may be suitable for use inperforming the invention. Such sequences may readily be used without anyundue experimentation.

[0132] Altering the expression of e gene can be the downregulation ofexpression. In the context of the current invention is envisaged thedownregulation of the expression of some proteins which are involved inthe process of processing of messenger RNA precursors.

[0133] This can for example result in a benificial effect on messengerRNA processing and stress tolerance, when said protein was a negativeregulator of the process. “Downregulation of expression” as used hereinmeans lowering levels of gene expression and/or levels of active geneproduct and/or levels of gene product activity. Decreases in expressionmay be accomplished by e.g. the addition of coding sequences or partsthereof in a sense orientation (if resulting in co-suppression) or in anantisense orientation relative to a promoter sequence and furthermore bye.g. insertion mutagenesis (e.g. T-DNA insertion or transposoninsertion) or by gene silencing strategies as described by e.g. Angelland Baulcombe 1998 (WO9836083), Lowe et al. 1989 (WO9853083), Lederer etal. 1999 (WO9915682) or Wang et al. 1999 (WO9953050). Genetic constructsaimed at silencing gene expression may have the nucleotide sequence ofsaid gene (or one or more parts thereof) contained therein in a senseand/or antisense orientation relative to the promoter sequence. Anothermethod to downregulate gene expression comprises the use of ribozymes,e.g. as described in Atkins et al. 1994 (WO9400012), Lenee et al. 1995(WO9503404), Lutziger et al. 2000 (WO00009619), Prinsen et al. 1997(WO9713865) and Scott et al. 1997 (WO9738116).

[0134] Modulating, including lowering, the level of active gene productsor of gene product activity can be achieved by administering or exposingcells, tissues, organs or organisms to said gene product, a homologue,analogue, derivative and/or immunologically active fragment thereof.Immunomodulation is another example of a technique capable ofdownregulation levels of active gene product and/or of gene productactivity and comprises administration of or exposing to or expressingantibodies to said gene product to or in cells, tissues, organs ororganisms wherein levels of said gene product and/or gene productactivity are to be modulated. Such antibodies comprise “plantibodies”,single chain antibodies, IgG antibodies and heavy chain camel antibodiesas well as fragments thereof. Within the scope of the present inventionare antibodies, recognizing the proteins of the present invention andthat can be used for said immunomodulation.

[0135] Modulating, including lowering, the level of active gene productsor of gene product activity can furthermore be achieved by administeringor exposing cells, tissues, organs or organisms to an inhibitor oractivator of said gene product or the activity thereof. Such inhibitorsor activators include proteins (comprising e.g. proteinases and kinases)and chemical compounds identified according to the current

[0136] By “cell fate and/or plant development and/or plant morphologyand/or biochemistry and/or physiology” is meant that one or moredevelopmental and/or morphological and/or biochemical and/orphysiological characteristics of a plant is altered by the performanceof one or more steps pertaining to the invention described herein.

[0137] “Cell fate” refers to the cell-type or cellular characteristicsof a particular cell that are produced during plant development or acellular process therefor, in particular during the cell cycle or as aconsequence of a cell cycle process.

[0138] “Plant development” or the term “plant developmentalcharacteristic” or similar term shall, when used herein, be taken tomean any cellular process of a plant that is involved in determining thedevelopmental fate of a plant cell, in particular the specific tissue ororgan type into which a progenitor cell will develop. Cellular processesrelevant to plant development will be known to those skilled in the art.Such processes include, for example, morphogenesis, photomorphogenesis,shoot development, root development, vegetative development,reproductive development, stem elongation, flowering, and regulatorymechanisms involved in determining cell fate, in particular a process orregulatory process involving the cell cycle.

[0139] “Plant morphology” or the term “plant morphologicalcharacteristic” or similar term will, when used herein, be understood bythose skilled in the art to refer to the external appearance of a plant,including any one or more structural features or combination ofstructural features thereof. Such structural features include the shape,size, number, position, colour, texture, arrangement, and patternationof any cell, tissue or organ or groups of cells, tissues or organs of aplant, including the root, stem, leaf, shoot, petiole, trichome, flower,petal, stigma, style, stamen, pollen, ovule, seed, embryo, endosperm,seed coat, aleurone, fibre, fruit, cambium, wood, heartwood, parenchyma,aerenchyma, sieve element, phloem or vascular tissue, amongst others.

[0140] “Plant biochemistry” or the term “plant biochemicalcharacteristic” or similar term will, when used herein, be understood bythose skilled in the art to refer to the metabolic and catalyticprocesses of a plant, including primary and secondary metabolism and theproducts thereof, including any small molecules, macromolecules orchemical compounds, such as but not limited to starches, sugars,proteins, peptides, enzymes, hormones, growth factors, nucleic acidmolecules, celluloses, hemicelluloses, calloses, lectins, fibres,pigments such as anthocyanins, vitamins, minerals, micronutrients, ormacronutrients, that are produced by plants.

[0141] “Plant physiology” or the term “plant physiologicalcharacteristic” or similar term will, when used herein, be understood torefer to the functional processes of a plant, including developmentalprocesses such as growth, expansion and differentiation, sexualdevelopment, sexual reproduction, seed set, seed development, grainfilling, asexual reproduction, cell division, dormancy, germination,light adaptation, photosynthesis, leaf expansion, fiber production,secondary growth or wood production, amongst others; responses of aplant to externally-applied factors such as metals, chemicals, hormones,growth factors, environment and environmental stress factors (eg.anoxia, hypoxia, high temperature, low temperature, dehydration, light,day length, flooding, salt, heavy metals, amongst others), includingadaptive responses of plants to said externally-applied factors.

[0142] The term “environmental stress” has been defined in differentways in the prior art and largely overlaps with the term “osmoticstress”. (Holmberg & Bülow, 1998, Trends plant sci. 3, 61-66) forinstance define different environmental stress factors which result inabiotic stress. The term osmotic stress as used herein is meant as astress situation induces by conditions as salinity, drought, heat,chilling (or cold) and freezing. With The term “environmental stress” asused in the present invention refers to any adverse effect onmetabolism, growth or viability of the cell, tissue, seed, organ orwhole plant which is produced by an non-living or non-biologicalenvironmental stress. More particularly, it also encompassesenvironmental factors such as water stress (flooding, water logging,drought, dehydration), anaerobic (low level of oxygen, CO₂ etc.),aerobic stress, osmotic stress, salt stress, temperature stress(hot/heat, cold, freezing, frost) or nutrients deprivation, pollutantsstress (heavy metals, toxic chemicals), ozone, high light, pathogen(including viruses, bacteria, fungi, insects and nematodes) andcombinations of these.

[0143] The term “anaerobic stress” means any reduction in oxygen levelssufficient to produce a stress as herein before defined, includinghypoxia and anoxia.

[0144] The term “flooding stress” refers to any stress which isassociated with or induced by prolonged or transient immersion of aplant, plant part, tissue or isolated cell in a liquid medium such asoccurs during monsoon, wet season, flash flooding or excessiveirrigation of plants, etc.

[0145] “Cold stress” or “chilling stress” and “heat stress” are stressesinduced by temperatures that are respectively, below or above, theoptimum range of growth temperatures for a particular plant species.Such optimum growth temperature ranges are readily determined or knownto those skilled in the art.

[0146] “Dehydration stress” is any stress which is associated with orinduced by the loss of water, reduced turgor or reduced water content ofa cell, tissue, organ or whole plant. “Drought stress” refers to anystress, which is induced by or associated with the deprivation of wateror reduced supply of water to a cell, tissue, organ or organism.

[0147] “Oxidative stress” refers to any stress, which increases theintracellular level of reactive oxygen species.

[0148] The terms “salinity-induced stress”, “salt stress” or “salt ionictoxicity” or mineral salt toxicity or similar terms refer to any stresswhich is associated with or induced by elevated concentrations of saltand which result in a perturbation in the osmotic potential of theintracellular or extracellular environment of a cell. The best knownexamples of mineral salts that induce said salt stress are Na+ and Li+.

[0149] The transgenic plants obtained in accordance with the method ofthe present invention, upon the presence of the polynucleic acid and/orregulatory sequence introduced into said plant, attain resistance,tolerance or improved tolerance or resistance against environmentalstress which the corresponding wild-type plant was susceptible to.

[0150] The terms “tolerance” and “resistance” cover the range ofprotection from a delay to complete inhibition of alteration in cellularmetabolism, reduced cell growth and/or cell death caused by theenvironmental stress conditions defined herein before. Preferably, thetransgenic plant obtained in accordance with the method of the presentinvention is tolerant or resistant to environmental stress conditions inthe sense that said plant is capable of growing substantially normalunder environmental conditions where the corresponding wild-type plantshows reduced growth, metabolism, viability, productivity and/or male orfemale sterility. As used herein, “stress tolerance” refers to thecapacity to grow and produce biomass during stress, the capacity toreinitiate growth and biomass production after stress, and the capacityto survive stress. The term “stress tolerance” also covers the capacityof the plant to undergo its developmental program during stresssimilarly to under non-stressed conditions, e.g. to switch from dormancyto germination and from vegetative to reproductive phase under stressedconditions similarly as under non-stressed conditions. Methodologies todetermine plant growth or response to stress include, but are notlimited to height measurements, leaf area, plant water relations,ability to flower, ability to generate progeny and yield or any othermethodology known to those skilled in the art.

[0151] “Growth” refers to the capacity of the plant or of plant parts togrow and increase in biomass while “yield” refers to the harvestablebiomass of plants or plant parts, particularly those parts of commercialvalue. “Growth and/or yield under stressed and non-stressed conditions”refers to the fact that field-grown plants almost always will experiencesome form of stress, albeit mild. It is therefore preferred not todistinguish non-stressed from mild-stressed conditions. As certainbeneficial effects of the invention on growth and yield are expected tooccur under both severe and mild stress conditions, they are thusdescribed as increasing growth andlor yield under stressed andnon-stressed conditions. Means for introducing recombinant DNA intoplant tissue or cells include, but are not limited to, transformationusing CaCl₂ and variations thereof, in particular the method describedpreviously (Hanahan 1983), direct DNA uptake into protoplasts (Krens etal. 1982; Paszkowski et al. 1984), PEG-mediated uptake to protoplasts(Armstrong et al. 1990) microparticle bombardment, electroporation(Fromm et al. 1985), microinjection of DNA (Crossway et al. 1986; Frommet al. 1985), microparticle bombardment of tissue explants or cells(Christou et al. 1988), vacuum-infiltration of tissue with nucleic acid,or in the case of plants, T-DNA-mediated transfer from Agrobacterum tothe plant tissue as described essentially (An et al 1985;Dodds1985;Herrera-Estrella et al. 1983a;Herrera-Estrella et al. 1983b).Methods for transformation of monocotyledonous plants are well known inthe art and include Agrobacterium-mediated transformation (Cheng et al.1997—WO9748814; Hansen 1998—WO9854961, Hiei et al. 1994—WO9400977;Hieiet al. 1998—WO9817813; Rikiishi et al. 1999—WO9904618; Saito et al.1995—WO9506722), microprojectile bombardment (Adams et al. 1999—U.S.Pat. No. 5,969,213; Bowen et al. 1998 —U.S. Pat. No. 5,736,369; Chang etal. 1994—WO9413822; Lundquist et al. 1999—U.S. Pat. No. 5,874,265/U.S.Pat No. 5,990,390; Vasil and Vasil 1995—U.S. Pat. No. 5,405,765; Walkeret al. 1999—U.S. Pat. No. 5,955,362), DNA uptake (Eyal et al.1993—W09318168), microinjection of Agrobacterium cells (von Holt1994—DE4309203) and sonication (Finer et al. 1997—U.S. Pat. No.5693512).

[0152] A whole plant may be regenerated from the transformed ortransfected cell, in accordance with procedures well known in the art.Plant tissue capable of subsequent clonal propagation, whether byorganogenesis or embryogenesis, may be transformed with a gene constructof the present invention and a whole plant regenerated therefrom. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, megagametophytes, callus tissue,existing meristematic tissue (e.g., apical meristem, axillary buds, androot meristems), and induced meristem tissue (e.g., cotyledon meristemand hypocotyl meristem).

[0153] Preferably, the plant is produced according to the inventivemethod is transfected or transformed with a genetic sequence, oramenable to the introduction of a protein, by any art-recognized means,such as microprojectile bombardment, microinjection,Agrobacterium-mediated transformation (including the “flower dip”transformation method; (Bechtold & Pelletier 1998;Trieu et al. 2000)),protoplast fusion, or electroporation, amongst others. Most preferablysaid plant is produced by Agrobacteriuim-mediated transformation.

[0154] With “binary transformation vector” is meant a T-DNAtransformation vector comprising: a T-DNA region comprising at least onegene of interest and/or at least one selectable marker active in theeukaryotic cell to be transformed; and a vector backbone regioncomprising at least origins of replication active in E. coli andAgrobacterium and markers for selection in E. coli and Agrobacterium.Alternatively, replication of the binary transformation vector inAgrobacterium is dependent on the presence of a separate helper plasmid.The binary vector pGreen and the helper plasmid pSoup form an example ofsuch a system as described in e.g. (Hellens et al. 2000) or as availableon the internet site http://www.pgreen.ac.uk.

[0155] The T-DNA borders of a binary transformation vector can bederived from octopine-type or nopaline-type Ti plasmids or from both.The T-DNA of a binary vector is only transferred to a eukaryotic cell inconjunction with a helper plasmid. Also known in the art are multiplebinary vector Agrobacterium strains for efficient co-transformation ofplants (Bidney and Scelonge 2000—W0001 8939).

[0156] “Host” or “host cell” or host organism” herein is any prokaryoticor eukaryotic cell or organism that can be a recipient of the sequencesof the present invention. A “host,” as the term is used herein, includesprokaryotic or eukaryotic organisms that can be genetically engineered.For examples of such hosts, see Maniatis et al., Molecular Cloning. ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1982). “plant cell” compnses any cell derived from any plant andexisting in culture as a single cell, a group of cells or a callus. Aplant cell may also be any cell in a developing or mature plant inculture or growing in nature.

[0157] “Plant” or “Plants” comprise all plant species which belong tothe superfamily Viridiplantae. The present invention is applicable toany plant, in particular a monocotyledonous plants and dicotyledonousplants including a fodder or forage legume, ornamental plant, food crop,tree, or shrub selected from the list comprising Acacia spp., Acer spp.,Actinidia spp.,Aesculus spp., Agathis australis, Albizia amara,Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Asteliafragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassicaspp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadabafarinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicumspp., Cassia spp., Centroema pubescens, Chaenomeles spp., Cinnamomumcassia, Coffea arabica, Colophospermum mopane, Coronillia varia,Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp.,Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogonspp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davalliadivaricata, Desmodium spp., Dicksonia squarosa, Diheteropogonamplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloapyramidalis, Ehrartia spp., Eleusine coracana, Eragrestis spp.,Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia villosa,Fagopyrum spp., Feijoa sellowiana, Fragaria spp., Flemingia spp,Freycinetia banksii, Geranium thunbergii, Ginkgo biloba, Glycinejavanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtiacoleosperma, Hedysarum spp., Hemarthia altissima, Heteropogon contortus,Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypertheliadissoluta, Indigo incarnata, Iris spp., Leptarrhena pyrolifolia,Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex,Lotonus bainesii, Lotus spp., Macrotyloma axillare, Malus spp., Manihotesculenta, Medicago sativa, Metasequoia glyptostroboides, Musasapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryzaspp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petuniaspp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photiniaspp., Picea glauca, Pinus spp., Pisum sativum, Podocarpus totara,Pogonarthria fleckii, Pogonarthria squarrosa, Populus spp., Prosopiscineraria, Pseudotsuga menziesii, Pterolbium stellatum, Pyrus communis,Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhusnatalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosaspp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitysverticillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghumbicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides,Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themedatriandra, Trifollum spp., Triticum spp., Tsuga heterophylla, Vacciniumspp., Vicia spp. Vitis vinifera, Watsonia pyramidata, Zantedeschiaaethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, brusselsprout, cabbage, canola, carrot, cauliflower, celery, collard greens,flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean,straw, sugarbeet, sugar cane, sunflower, tomato, squash, and tea,amongst others, or the seeds of any plant specifically named above or atissue, cell or organ culture of any of the above species The presentinvention is applicable to any plant, in particular a monocotyledonousplants and dicotyledonous plants including a fodder or forage legume,ornamental plant, food crop, tree, or shrub selected from the listcomprising Acacia spp., Acer. spp., Actinidia spp.,Aesculus spp.,Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp.,Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaeaplurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkeaafricana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camelliasinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens,Chaenomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermummopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumisspp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeriajaponica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergiamonetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa,Diheteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum,Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana, Eragrestisspp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulaliavillosa, Fagopyrum spp., Feijoa sellowiana, Fragaria spp., Flemingiaspp, Freycinetia banksii, Geranium thunbergii, Ginkgo biloba, Glycinejavanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtiacoleosperma, Hedysarum spp., Hemarthia altissima, Heteropogon contortus,Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypertheliadissoluta, Indigo incamata, Iris spp., Leptarrhena pyrolifolia,Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex,Lotonus bainesai, Lotus spp., Macrotyloma axillare, Malus spp., Manihotesculenta, Medicago sativa, Metasequoia glyptostroboides, Musasapientum, Nicotianum spp.,

[0158] Onobrychis spp., Omithopus spp., Oryza spp., Peltophorumafricanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolusspp., Phoenix canariensis, Phormium cookianum, Photinia spp., Piceaglauca, Pinus spp., Pisum sativum, Podocarpus totara, Pogonarthriafleckii, Pogonarthria squarrosa, Populus spp., Prosopis cineraria,Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercusspp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis,Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubusspp., Salix spp., Schyzachynum sanguineum, Sciadopitys verticillata,Sequoia sempemrens, Sequoiadendron giganteum, Sorghum bicolor, Spinaciaspp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthoshumilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifoliumspp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp.Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays,amaranth, artichoke, asparagus, broccoli, brussel sprout, cabbage,canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil,oilseed rape, okra, onion, potato, rice, soybean, straw, sugarbeet,sugar cane, sunflower, tomato, squash, and tea, amongst others, or theseeds of any plant specifically named above or a tissue, cell or organculture of any of the above species “Cereal” comprises crop plants withedible grain for example plants belonging to the grass family that iscultivated for its nutritious grains such as oats, barley, rye, wheat,rice, and corn etc.

[0159] Within the scope of the present invention is also the applicationof the Two-hybrid system, wherein any of the sequences of the presentinvention are used to study interactions with other factors such asproteins. With “yeast two-hybrid assay” is meant an assay that is basedon the observation that many eukaryotic transcription factors comprisetwo domains, a DNA-binding domain (DB) and an activation domain (AD)which, when physically separated (i.e. disruption of the covalentlinkage) do not effectuate target gene expression. Two proteins able tointeract physically with one of said proteins fused to DB and the otherof said proteins fused to AD will re-unite the DB and AD domains of thetranscription factor resulting in target gene expression. The targetgene in the yeast two-hybrid assay is usually a reporter gene such asthe β-galactosidase gene. Interaction between protein partners in theyeast two-hybrid assay can thus be quantified by measuring the activityof the reporter gene product (Bartel & Fields 1997). Alternatively, amammalian two-hybrid system can be used which includes e.g. a chimericgreen fluorescent protein encoding reporter gene (Shioda et al. 2000).Yet another alternative consists of a bacterial two-hybrid system usinge.g. HIS as reporter gene (Joung et al. 2000). A person skilled in theart will also recognise that in adapted versions of the two-hybridsystem and also other techniques (e.g. gel-retardation,immunoprecipitation, competitive inhibition), it is possible to studyprotein-oligonucleotide interactions, and therefor these techniqueswhich use any of the sequences of the present invention are also withinthe scope of the invention.

[0160] The term “fragment of a sequence” or “part of a sequence” means atruncated sequence of the original sequence referred to. The truncatedsequence (nucleic acid or protein sequence) can vary widely in length;the minimum size being a sequence of sufficient size to provide asequence with at least a comparable function and/or activity or theoriginal sequence referred to, while the maximum size is not critical.In some applications, the maximum size usually is not substantiallygreater than that required to provide the desired activity and/orfunction(s) of the original sequence. Typically, the truncated aminoacid or nucleotide sequence will range from about 5 to about 60 aminoacids in length. More typically, however, the sequence will be a maximumof about 50 amino acids in length, preferably a maximum of about 60amino acids. It is usually desirable to select sequences of at leastabout 10, 12 or 15 amino acids or nucleotides, up to a maximum of about20 or 25 amino acids or nucleotides.

[0161] The invention also includes methods for high throughput compoundscreening. The man skilled in the art can easily design such methodsusing one or several elements of the invention.

[0162] The compounds yet to be obtained or identified can be compoundsthat are able to bind to any of the nucleic acids, peptides or proteinsinvolved in the process of processing precursor messenger RNA and aretherefore useful in the method of the present invention. Said compoundor plurality of compounds may be comprised in, for example, samples,e.g., cell extracts from, e.g., plants, animals or micro-organisms.Furthermore, said compound(s) may be known in the art but hitherto notknown to be capable of suppressing or activating proteins involved inprocessing of precursor messenger RNA.

[0163] In the scope of the present invention is also included theintroduction into a plant cell one or more recombinant nucleic acidmolecules, such as a DNA molecule encoding a protein which whenexpressed in said plant cell at an effective amount increases or inducesthe expression of an endogenous polynucleotide acid according to thepresent invention or as defined in claims or increases or induces theactivity of a polypeptide of claim.

[0164] The present invention is further described by reference to thefollowing non-limiting figures and examples.

SHORT DESCRIPTION OF THE FIGURES

[0165]FIG. 1

[0166] Salt-tolerance phenotypes of yeast strains expressing theisolated Arabidopsis cDNA clones, U1A, Ct-SRL1 or RCY1, as determined by“drop-tests”; in which serial dilutions of saturated cultures of thedifferent strains, and of the control strain containing the emptyexpression vector, are tested on plates, without salt or containing theindicated LiCl or NaCl concentrations.

[0167]FIG. 2

[0168] Amino acid sequences derived from the nucleotide sequences ofclones RCY1 (A: SEQ ID NO 4) and Ct-SRL1 (B: SEQ ID NO 3). DipeptidesArg-Ser. Arg-Glu and Arg-Asp. which define the RS domain, are written inbold letters. The underlined M in (A) marks the methionine residue usedas initiator during expression in yeast of the RS domain of RCY1 (SEQ IDNO 21).

[0169]FIG. 3

[0170] Salt-tolerance phenotype of yeast strains expressing theamino-terminal (cyclin) domain of RCY1, its carboxy-terminal RS domain,or the full-length protein. Drop-tests were performed as in theexperiment of FIG. 1.

[0171]FIG. 4

[0172] Salt-tolerance phenotype of transgenic Arabidopsis plants. T2seeds from three independent transgenic lines (L1, L3 and L5, as anexample of the 12 obtained lines), transformed with the Ct-SRL 1 cDNAunder control of the 35S promoter from CaMV, were germinated on agarplates without (A) or with (B) 20mM LiCl. Seeds from wild-type plants(Wt) were used as control. All transgenic lines showed similarphenotypes.

[0173]FIG. 5

[0174] Sequences of the genes and proteins of the present invention.

EXAMPLES Example 1 Plant Material

[0175] Seeds of the red beet (Beta vulgaris var. DITA, also referred toherein as “sugar beet”), were sown on pots containing a mixture of sandand vermiculite (1:1 w/w). The plants were grown under greenhouseconditions (8 hours at 20° C., 16 hours at 25° C. with supplementarylighting to stimulate a minimum of 12 hours photoperiod). They wereperiodically irrigated with a nutrient solution containing 2.4 g/lCa(NO₃)₂.4H₂O, 1 g/l KNO₃, 1 g/l MgSO₄.7H₂O, 0.3 g/l KH₂PO₄, 5.6 mg/lFe-quelate (Kelantren, Bayer), 1.1 mg/l ZnSO₄.7H₂O, 3.3 mg/l MnO₄.H₂O,0.3 mg/l CuSO₄.5H₂O, 3.8 mg/l H₃BO₃, 0.18 mg/l (NH₄)6Mo₇.4H₂O. For theconstruction of the cDNA library, three-week-old plants were irrigatedwith 200 mM NaCl for 24 hours before harvesting.

Example 2 Yeast Strains and Culture Conditions

[0176] The Saccharomyces cerevisiae competent cells W303-1A (MATa ura3,leu2, his3, trp1, ade2, ena 1-4::HIS3) were transformed with theArabidopsis cDNA library. The Saccharomyces cerevisiae strain JM26 (MATaleu 2-3,112 ura 3-1 trp1-1, ade 2-1 his3-11,15 can 1-100, ena 1-4::HIS3,nha1:TRP1) provided by J. M. Mulet (Universidad Politécnica de Valencia,Instituto de Biologia Molecular y Cellular de Plantas) was used for thescreening of the red beet cDNA library. Strain JM26 is a derivative ofW303.1 A (Wallis et al., 1989, Cell 58: 409-419) with null mutations ofthe genes ENA1-4 and NHA1, encoding a Na⁺-pumping ATPase and aNa⁺/H⁺antiporter, respectively, responsible for most of the yeast sodiumextrusion (Garciadeblas et al. 1993, Mol. Gen. Genet. 236, 363-368),(Bañuelos et al. 1998, Microbiology 144: 2749-2758).

[0177] The yeast cells were grown in either minimal synthetic glucosemedium (SD) or rich medium (YPD). SD medium contained 2% glucose, 0.7%yeast nitrogen base without amino acids and 50 mM succinic acid,adjusted to pH 5 with Tris, plus the required amino acids [100 μg/mlleucine, 30 μg/ml adenine, 100 μg/ml methionine] as indicated. YPDmedium contained 1% yeast extract, 2% Bacto peptone and 2% glucose.Media were supplemented with NaCl and LiCl as indicated in the figuresand the examples. Solid media contained 2% bacteriological-grade agar.

Example 3 Construction of the cDNA Libraries

[0178] The construction of the Arabidopsis thaliana cDNA library in thevector pFL61 is described in Minet et al. (1992) Plant J. 2(3): 417-422.

[0179] For the construction of a red beet cDNA library induced by saltstress, the plant material as described in example 1 was used.Directional cDNAs were synthesised (cDNA synthesis kit, Stratagene)using poly(A)⁺ RNA prepared from leaves of salt-treated red beet plants.cDNAs were ligated into phage λPG15 vector and packaged using a GigapackIII gold packaging extract (Stratagene). This phage has inserted theexcisable expression plasmid pYPGE15 (URA3 as a selection marker) thatis usable directly for both E. coli and yeast complementation (Brunelliand Pall, 1993, Yeast 9: 1309-1318). A plasmid cDNA library wasrecovered from λPG15 by the cre-lox recombinase system (Brunelli andPall, 1993, Yeast 9: 1309-1318).

Example 4 Screening and Isolation of cDNA Clones Conferring SaltTolerance to Yeast

[0180] To screen for Arabidopsis thaliana cDNAs which increase salttolerance in yeast, the cDNA library constructed in pFL61 was used totransform the yeast strain W303-1A by the LiCl method (Gietz et al.1992, Nucleic Acids Res. 20: 1425). Transformants were screened forhalotolerance in plates with minimal medium plus 25 mM and 50 mM LiCl,or as indicated in the figures, and containing 400 μM methionine.Resistant clones were subjected to fluoroorotic acid-induced plasmidloss (Boeke et al (1984) Mol. Gen. Genet. 197: 354-346) to select onlythose clones showing plasmid dependent LiCl tolerance. Results wereconfirmed in wild type strain and in the double mutant ena1-4::HIS3tfp1::LEU2, defective in the vacuolar transport.

[0181] To screen for red beet cDNAs which increase salt tolerance inyeast, the cDNA library constructed in pYPGE15 was used to transform theyeast mutant strain JM26. Transformants selected on SD plates withleucine and adenine by uracil prototrophy were pooled and replated onscreening medium (SD with leucine, adenine and methionine supplementedwith 0.15 M NaCl) at a density of 2×10⁵ cells per plate (12×12 cm).Methionine was added to the selective medium to avoid selection of theHAL2-like homologues already found in Arabidopsis (Quintero et al. 1996,Plant Cell 8: 529-537) (Gil-Mascarell et al. 1999, Plant J. 17(4):373-383). Alternatively, for the selection of Li+resistant yeast cells,the transformants were replated on screening medium (SD with leucine andadenin supplemented with 20 mM LiCl). The putative positive clones wererescreened on the same NaCl or LiCl medium.

Example 5 Determination of Intracellular Lithium Content

[0182] Yeast cells expressing the cDNA clones of the present inventionor the control strain transformed with the empty vector, were grown toexponential phase and the medium was then supplemented with LiCl to 30mM final concentration. 10 ml—samples were taken at different times andthe intracellular lithium content was determined as described previously(Murgùia et al. 1996, J. Biol. Chem. 271: 29029-29033).

Example 6 Beta Galactosidase Assay

[0183] Yeast cells containing the E. Coli LacZ gene, interrupted or notwith an intron (Legrain and Rosbash (1989), cell, 75: 573-583), undercontrol of an galactose-inducible promoter, were transformed with theCt-SRL1 cDNA in a yeast expression vector or, as a control, with theempty plasmid. Cultures were grown to exponential phase in glucoseminimal medium, with or without 35 mM LCl, and then shifted to galactosemedium, maintaining the same salt conditions, to induce thebeta-galactosidase expression. Samples were collected at 0 (backgroundvalue) and 4 hours after induction, and beta-galactosidase activity wasmeasured in permeabilised cells as described (Serrano et al, 1973: Eur.J. Biochem. 34: 479-482).

Example 7 RT-PCR Assay

[0184] Yeast cells overexpressing the Ct-SRL cDNA or transformed withthe empty vector were grown to exponential phase; the medium was thensupplemented with LiCl to 150 mM final concentration and total RNA waspurified at different times. Equal amounts of total RNA were digestedwith RNase-free DNasel, and reverse-transcribed with M-MuLV RTase (RocheMolecular Biochemicals) using primer preSARlrp (5′ CATCAAATCTTTCAGGG-3′:SEQ ID NO. 23), that hybridises to the second SAR1 exon 277 nucleotidesdownstream from the 3′-splice site. It was followed by 15 cycles of PCRamplification with Netzyme (Molecular Netline Bioproducts) DNApolymerase, and primer preSAR1fp, (5′-CTTTATTTTACTGTACAG-3′: SEQ ID NO.24), which hybridises to the 3′ end of the SAR1 intron. [α-³²P]dCTP(740kBq, 110 TBq/mmol, Amersham) was added after the 5^(th) cycle andthe products were resolved in 6% PA-urea gels and visualised byautoradiography. The relative intensity of the amplified bands wasdetermined using a FUJI (Fujifilm BAS-1500) phosphorimager. Sampleslacking reverse transcriptase ware used as controls.

Example 8 Transformation of Plants

[0185] The clones of the present invention, for example the ArabidopsisCt-SRL1 cDNA, are subcloned into pBI121 (Clontech), in place of the GUSgene, and the resulting binary vector is introduced into theAgrobacterium tumefaciens strain C58C1 by electroporation. Transgenicplants, like Arabidopsis plants were obtained by in vivo infiltration asdescribed in http/www.arabidopsis.org/protocols_Mundy2.html#trans.inf.

[0186] Alternatively, the genes of the present invention can betransformed to other dicotyledon or monocotyledon plants. Therefore theyare cloned in the suitable plant transformation vector, such as a binaryvector, under the control of plant operable regulatory sequences, suchas a plant operable promoter and a plant operable terminator. Thesevectors comprising a gene of the present invention can be transformedinto plants (such as a crop plant) using standard techniques well knownby the person skilled in the art, such as agrobacterium mediated genetransfer. The transgenic plant expressing the gene of the presentinvention are expected to show an increased tolerance to environmentalstress, such as for example an increased tolerance to mineral salttoxicity. This increased tolerance can for example be observed by theability of the transgenic plants to germinate in salt containing medium.

Example 9 Rice Transformation with the Genes of the Present Invention

[0187] The expression of red beet genes or Arabidopsis thaliana that areinvolved in salt tolerance in yeast in monocotyledons, such as forexample Rice, can confer stress tolerance to that monocotyledoneousplant.

[0188] To transfer the stress tolerance activity of the genes of thepresent invention to monocots, the aforementioned genes (SEQ ID NO. 1,2, 5, 7, 9, 11, 13, 15, 17 and 19), operably linked to a promoter, areeach transformed to rice using the standard transformation procedureswell known to the persons skilled in the art and outlined in thefollowing paragraph. After several time periods ranging from 1 day to 1or more weeks, the seedling is checked for the expression of thetransformed gene. This is done by growing the seedlings in organogenesismedium, and checking the presence of the DNA or mRNA by PCR or reversePCR. After the confirmation of gene expression the transformed riceplants are checked for the enhanced tolerance to stress situationsincluding salt, drought and cold (see W097/13843). This is done bygrowing the transformed rice plants in medium containing increasedamounts of NaCl or LiCl. Also the increased resistance to cold ordrought is tested by growing the transformed plants in suboptimalgrowing temperatures and suboptimal levels of humidity, respectively(WO97/13843).

[0189] Agrobacterium-Mediated Rice Transformation

[0190] The genes of the present invention are operably linked to apromoter and cloned into a vector. These vectors are transformed toAgrobacterium tumefaciens strain LBA4404 or C58 by means ofelectroporation and subsequently transformed bacterial cells areselected on a solid agar medium containing.the appropriate antibiotics.

[0191] For demonstration of the expression of the genes of the currentinvention in rice, 309 mature dry seeds of the rice japonica cultivarsNipponbare or Taipei are dehusked, sterilised and germinated on a mediumcontaining 2,4-dichlorophenoxyacetic acid (2,4-D). After incubation inthe dark for four weeks, embryogenic, scutellum-derived calli areexcised and propagated on the same medium. Selected embryogenic callusis then co-cultivated with Agrobacterium. Co-cultivated callus is grownon 2,4D-containing medium for 4 to 5 weeks in the dark in the presenceof a suitable concentration of the appropriate selective agent. Duringthis period, rapidly growing resistant callus islands develop. Aftertransfer of this material to a medium with a reduced concentration of2,4-D and incubation in the light, the embryogenic potential is releasedand shoots develop in the next four to five weeks. Shoots are excisedfrom the callus and incubated for one week on an auxin-containing mediumfrom which they can be transferred to the soil. Hardened shoots aregrown under high humidity and short days in a phytotron. Seeds can beharvested three to five months after transplanting. The method yieldssingle locus transformants at a rate of over 50% (Chan et al. 1993,Plant Mol. Biol. 22 :491-506) (Hiei et al. 1994, Plant J. 6: 271-282)

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1 24 1 840 DNA Arabidopsis thaliana 1 caaaccttga gaagatgaat ttaccaactaaaccttctgg ttcaaccgga gacatgaccc 60 gtggctcaga agacactgcc cgtcgtccaccatcagtaaa agcatctctc tctgtttcat 120 ttggtcagcg tgcacctcat cgtgcttccaccagaggctc ttctcctgtt cgccgtcctc 180 caccgactgg ttatgacaga aatggaggcgatgaagtaca acagcggtcc ccacgtagaa 240 gccagagccg agactattat tctgacagagactcagatag acaacgggaa agagagaggg 300 agaaagaccg cgaaagagag agggggagggatagatacag agaaagggag agggattatg 360 gtaatgatag gagatcaagg cgcgactatgatagtagaag caggcgcaat gattatgagg 420 acgacagaag tagacatgac cggagaagcaggagcagaag cagaagtagg agcaggagtg 480 tgcagattga gcgtgaaccg actcctaaaagagatagtag caacaaagag aaatcggcgg 540 tgacagtgaa cagcaatctc gcaaagctaaaagatttgta tggagacgca agtagtcaga 600 aaagggatga aggatttgga acaaggaaagattcaagttc agaagaagtg ataaagcttg 660 gtggttcctc ttggaggtga aaaaacaaacaaaacaaaac caaactgtgg atttaaaatg 720 cttcttctat ttagcaggat gatgcatgttgtttaactat actgttttga tttccagcaa 780 actatttgtc atatgcttta tattacagtttaagagttga tctttatctt gaaaaaaaaa 840 2 1433 DNA Arabidopsis thaliana 2gtttagtgaa tcgatgattt acactgctat cgacaatttt tacctaaccg acgagcagct 60gaaggcttca ccttcgagga aagatgggat agatgaaaca actgaaatct ctcttagaat 120ctatggatgt gatctcatcc aagagggtgg aatattgctc aaactaccac aggcagttat 180ggctactggg caggttctgt ttcagcgatt ctattgcaag aagtctttgg ctaaatttga 240tgtcaagata gttgctgcca gctgtgtatg gcttgcatca aaactggaag aaaaccctaa 300aaaagctaga caggtcatca tcgtattcca caggatggag tgtcgcaggg agaacttgcc 360attagaacat ctggatatgt atgccaagaa gttctctgag ttgaaagttg aattaagcag 420aactgagaga catatactga aagagatggg ttttgtttgt catgttgaac atcctcacaa 480gttcatatca aactaccttg ccacattaga aacacctcca gaattgaggc aagaagcttg 540gaatttggcc aatgatagtc tgcgtacaac cctctgtgta aggttcagaa gtgaggttgt 600ggcttgtggg gtagtgtatg ctgctgcccg taggtttcaa gtaccactcc ctgagaatcc 660gccgtggtgg aaagcatttg atgcagataa atctagtatt gacgaagtgt gtagagttct 720tgctcattta tacagtcttc caaaggctca gtatatctct gtttgcaagg atgggaagcc 780atttacattt tctagcagat ccgggaattc tcaaggtcaa tcagcgacaa aggatctgtt 840gccgggagca ggcgaggctg ttgatactaa atgtactgca ggatcagcta ataacgactt 900gaaggatgga atggttacta caccacacga aaaggctaca gattccaaga aaagtggtac 960cgagtcaaac tctcagccaa ttgtaggaga ctcaagctat gaaagaagta aagtaggaga 1020tagagaaaga gagagtgata gagagaagga acgaggtaga gagagggaca ggggtaggtc 1080tcacagaggc agagattctg acagagacag tgatagggag agagacaaac tcaaagatcg 1140aagtcatcat cggtcaagag acagattgaa ggattcaggt ggacattcag ataaatcaag 1200gcatcattct tctcgggacc gtgactaccg cgactcatcg aaagaccgtc gtaggcacca 1260ttaagccaat cttcttgtca tctacatccc cttgagccta cttgatgtta agacagtata 1320gtgttgtatt gtgttaagag tcaaaaccca tgtgtactta atcacatgct aagatcacgt 1380tggttcgaca tataaatcga gaaagtctga tatgtttcta aaaaaaaaaa aaa 1433 3 221PRT Arabidopsis thaliana 3 Met Asn Leu Pro Thr Lys Pro Ser Gly Ser ThrGly Asp Met Thr Arg 1 5 10 15 Gly Ser Glu Asp Thr Ala Arg Arg Pro ProSer Val Lys Ala Ser Leu 20 25 30 Ser Val Ser Phe Gly Gln Arg Ala Pro HisArg Ala Ser Thr Arg Gly 35 40 45 Ser Ser Pro Val Arg Arg Pro Pro Pro ThrGly Tyr Asp Arg Asn Gly 50 55 60 Gly Asp Glu Val Gln Gln Arg Ser Pro ArgArg Ser Gln Ser Arg Asp 65 70 75 80 Tyr Tyr Ser Asp Arg Asp Ser Asp ArgGln Arg Glu Arg Glu Arg Glu 85 90 95 Lys Asp Arg Glu Arg Glu Arg Gly ArgAsp Arg Tyr Arg Glu Arg Glu 100 105 110 Arg Asp Tyr Gly Asn Asp Arg ArgSer Arg Arg Asp Tyr Asp Ser Arg 115 120 125 Ser Arg Arg Asn Asp Tyr GluAsp Asp Arg Ser Arg His Asp Arg Arg 130 135 140 Ser Arg Ser Arg Ser ArgSer Arg Ser Arg Ser Val Gln Ile Glu Arg 145 150 155 160 Glu Pro Thr ProLys Arg Asp Ser Ser Asn Lys Glu Lys Ser Ala Val 165 170 175 Thr Val AsnSer Asn Leu Ala Lys Leu Lys Asp Leu Tyr Gly Asp Ala 180 185 190 Ser SerGln Lys Arg Asp Glu Gly Phe Gly Thr Arg Lys Asp Ser Ser 195 200 205 SerGlu Glu Val Ile Lys Leu Gly Gly Ser Ser Trp Arg 210 215 220 4 416 PRTArabidopsis thaliana 4 Met Ile Tyr Thr Ala Ile Asp Asn Phe Tyr Leu ThrAsp Glu Gln Leu 1 5 10 15 Lys Ala Ser Pro Ser Arg Lys Asp Gly Ile AspGlu Thr Thr Glu Ile 20 25 30 Ser Leu Arg Ile Tyr Gly Cys Asp Leu Ile GlnGlu Gly Gly Ile Leu 35 40 45 Leu Lys Leu Pro Gln Ala Val Met Ala Thr GlyGln Val Leu Phe Gln 50 55 60 Arg Phe Tyr Cys Lys Lys Ser Leu Ala Lys PheAsp Val Lys Ile Val 65 70 75 80 Ala Ala Ser Cys Val Trp Leu Ala Ser LysLeu Glu Glu Asn Pro Lys 85 90 95 Lys Ala Arg Gln Val Ile Ile Val Phe HisArg Met Glu Cys Arg Arg 100 105 110 Glu Asn Leu Pro Leu Glu His Leu AspMet Tyr Ala Lys Lys Phe Ser 115 120 125 Glu Leu Lys Val Glu Leu Ser ArgThr Glu Arg His Ile Leu Lys Glu 130 135 140 Met Gly Phe Val Cys His ValGlu His Pro His Lys Phe Ile Ser Asn 145 150 155 160 Tyr Leu Ala Thr LeuGlu Thr Pro Pro Glu Leu Arg Gln Glu Ala Trp 165 170 175 Asn Leu Ala AsnAsp Ser Leu Arg Thr Thr Leu Cys Val Arg Phe Arg 180 185 190 Ser Glu ValVal Ala Cys Gly Val Val Tyr Ala Ala Ala Arg Arg Phe 195 200 205 Gln ValPro Leu Pro Glu Asn Pro Pro Trp Trp Lys Ala Phe Asp Ala 210 215 220 AspLys Ser Ser Ile Asp Glu Val Cys Arg Val Leu Ala His Leu Tyr 225 230 235240 Ser Leu Pro Lys Ala Gln Tyr Ile Ser Val Cys Lys Asp Gly Lys Pro 245250 255 Phe Thr Phe Ser Ser Arg Ser Gly Asn Ser Gln Gly Gln Ser Ala Thr260 265 270 Lys Asp Leu Leu Pro Gly Ala Gly Glu Ala Val Asp Thr Lys CysThr 275 280 285 Ala Gly Ser Ala Asn Asn Asp Leu Lys Asp Gly Met Val ThrThr Pro 290 295 300 His Glu Lys Ala Thr Asp Ser Lys Lys Ser Gly Thr GluSer Asn Ser 305 310 315 320 Gln Pro Ile Val Gly Asp Ser Ser Tyr Glu ArgSer Lys Val Gly Asp 325 330 335 Arg Glu Arg Glu Ser Asp Arg Glu Lys GluArg Gly Arg Glu Arg Asp 340 345 350 Arg Gly Arg Ser His Arg Gly Arg AspSer Asp Arg Asp Ser Asp Arg 355 360 365 Glu Arg Asp Lys Leu Lys Asp ArgSer His His Arg Ser Arg Asp Arg 370 375 380 Leu Lys Asp Ser Gly Gly HisSer Asp Lys Ser Arg His His Ser Ser 385 390 395 400 Arg Asp Arg Asp TyrArg Asp Ser Ser Lys Asp Arg Arg Arg His His 405 410 415 5 920 DNAArabidopsis thaliana 5 ccacgcgtcc gagagatttg gtgaagacga tggagatgcaagaggctaat caaggaggag 60 gatcggaggt ttctccgaat cagacgattt acatcaacaatctcaacgaa aaagtgaagc 120 ttgatgagct gaagaaatcg ctgaatgcag tgttctctcagttcgggaag atactggaga 180 tattggcgtt taagaccttt aagcacaaag gacaagcttgggtagtcttc gacaacaccg 240 agtctgcttc cactgctatt gctaaaatga ataattttcctttctacgac aaggagatga 300 gaatacaata tgccaaaaca aaatcagatg ttgttgccaaggccgatggt acatttgttc 360 ctcgcgagaa gagaaagaga catgaggaga aaggaggcggcaagaaaaag aaagaccagc 420 accatgattc tacacagatg ggcatgccca tgaactcagcatatccaggt gtctatggag 480 ctgcacctcc tctatcgcaa gtaccatacc ctggtggcatgaaacccaat atgcccgagg 540 caccagctcc gccaaataat attctctttg tccaaaaccttcctcacgag acaactccaa 600 tggtgcttca gatgttgttc tgccagtacc aaggatttaaggaagttaga atgattgaag 660 ccaaaccggg aatcgccttt gtggagtttg ctgatgagatgcagtcgacg gtcgcaatgc 720 agggacttca aggtttcaag attcagcaaa accagatgctcatcacgtat gccaagaaat 780 agacaatttc gttttatttg tgtttcgatg agatatgtttgtatctgtca atgttacttc 840 ttgccatggg ggctgtcttc tgggttgtgt gatgctagatatccctctct acttacattt 900 tttcatcaaa aaaaaaaaaa 920 6 250 PRTArabidopsis thaliana 6 Met Glu Met Gln Glu Ala Asn Gln Gly Gly Gly SerGlu Val Ser Pro 1 5 10 15 Asn Gln Thr Ile Tyr Ile Asn Asn Leu Asn GluLys Val Lys Leu Asp 20 25 30 Glu Leu Lys Lys Ser Leu Asn Ala Val Phe SerGln Phe Gly Lys Ile 35 40 45 Leu Glu Ile Leu Ala Phe Lys Thr Phe Lys HisLys Gly Gln Ala Trp 50 55 60 Val Val Phe Asp Asn Thr Glu Ser Ala Ser ThrAla Ile Ala Lys Met 65 70 75 80 Asn Asn Phe Pro Phe Tyr Asp Lys Glu MetArg Ile Gln Tyr Ala Lys 85 90 95 Thr Lys Ser Asp Val Val Ala Lys Ala AspGly Thr Phe Val Pro Arg 100 105 110 Glu Lys Arg Lys Arg His Glu Glu LysGly Gly Gly Lys Lys Lys Lys 115 120 125 Asp Gln His His Asp Ser Thr GlnMet Gly Met Pro Met Asn Ser Ala 130 135 140 Tyr Pro Gly Val Tyr Gly AlaAla Pro Pro Leu Ser Gln Val Pro Tyr 145 150 155 160 Pro Gly Gly Met LysPro Asn Met Pro Glu Ala Pro Ala Pro Pro Asn 165 170 175 Asn Ile Leu PheVal Gln Asn Leu Pro His Glu Thr Thr Pro Met Val 180 185 190 Leu Gln MetLeu Phe Cys Gln Tyr Gln Gly Phe Lys Glu Val Arg Met 195 200 205 Ile GluAla Lys Pro Gly Ile Ala Phe Val Glu Phe Ala Asp Glu Met 210 215 220 GlnSer Thr Val Ala Met Gln Gly Leu Gln Gly Phe Lys Ile Gln Gln 225 230 235240 Asn Gln Met Leu Ile Thr Tyr Ala Lys Lys 245 250 7 1492 DNA Betavulgaris 7 tcctctcttt taacctaatt tgagctctca ttatcctgat tttttcaaccatggatgctc 60 agagagctct tctcgatgaa ttaatgggcg cagctcgaaa tctgactgatgaagaaaaga 120 aaggttatag agagataaag tgggatgaca aggaagtttg tgcgccgtatatgattcgat 180 tttgccctca cgatctcttc gtcaatactc gaagtgatct tggaccatgtccaagagttc 240 atgaccaaaa gctgaaagag agctttgaga actctccaag gcatgactcatatgtcccac 300 gttttgaagc agagcttgcc caattttgtg agaagctggt ggcagatttggataggaaag 360 taagacgtgg gagagagcgg ctggaccagg aggttgaacc tccacctccccctcctattt 420 ctgcagaaaa agctgagcag ctatctgtac ttgaagagaa aataaaaaatttgcttgaac 480 aagtagagtc actgggagaa gctggcaaag tcgatgaagc agaagcactcatgcgaaagg 540 tggaaagtct taatttagag aaagctgcat taactcaaca gccccagaatgcagcaacaa 600 tgcttaccca agagaaaaag atggcactat gtgaaatttg cggttccttcctggtagcca 660 atgatgctgt ggaaagaact caatctcata taactggcaa gcagcatattggctatggca 720 tggtccgtga ttaccttgct gagtataagg aggctaagga gaaggcaagagaagaggaaa 780 gattagcaag ggagaaagaa gcagaagaac gtcggaagca gagggaaaaggaaaatgaga 840 gtaaaaacag aagaagcatc tccagtgaga gggaccgtca tcgtgatagggattatggcc 900 gagatcgtga aagatcacga gaatggaaca atagggggaa tcgagacgagggaagaggaa 960 tggatcggag aaggcaatat gatcgcaatg gaagggatgg agggaggaatacgtatcatg 1020 gtcgtgaacg tgaaaggagc aggtcacggt cccctgttag gcatggccaccggaggtgat 1080 ctaagagtgc tggttgccga tattagtagg cagtgggttg tgtagataaacgatgatctt 1140 aaacctactg aggtagatgc tttatatctc aagatgtttt gtgtctgttttcgaggtgtt 1200 gcattgcagt cttattgggg gttaaacttt tctttattgt cccacagtgttgagactata 1260 ctgtctcctc tcatcaatct tgttagaggt caaagagatt gaggtaggtaaaacttcatc 1320 gttgtaatct tacctatagt caacttgagt tttgtccaat tatagcacatggtctttgaa 1380 acatttttta atcatgcggg ggtacgcaag aaaatatgca actatgctgcatggcttgtg 1440 tgcaaaaaaa aaaaaaaaaa aaaaaactcg agggggggcc cggtaccaagat 1492 8 342 PRT Beta vulgaris 8 Met Asp Ala Gln Arg Ala Leu Leu AspGlu Leu Met Gly Ala Ala Arg 1 5 10 15 Asn Leu Thr Asp Glu Glu Lys LysGly Tyr Arg Glu Ile Lys Trp Asp 20 25 30 Asp Lys Glu Val Cys Ala Pro TyrMet Ile Arg Phe Cys Pro His Asp 35 40 45 Leu Phe Val Asn Thr Arg Ser AspLeu Gly Pro Cys Pro Arg Val His 50 55 60 Asp Gln Lys Leu Lys Glu Ser PheGlu Asn Ser Pro Arg His Asp Ser 65 70 75 80 Tyr Val Pro Arg Phe Glu AlaGlu Leu Ala Gln Phe Cys Glu Lys Leu 85 90 95 Val Ala Asp Leu Asp Arg LysVal Arg Arg Gly Arg Glu Arg Leu Asp 100 105 110 Gln Glu Val Glu Pro ProPro Pro Pro Pro Ile Ser Ala Glu Lys Ala 115 120 125 Glu Gln Leu Ser ValLeu Glu Glu Lys Ile Lys Asn Leu Leu Glu Gln 130 135 140 Val Glu Ser LeuGly Glu Ala Gly Lys Val Asp Glu Ala Glu Ala Leu 145 150 155 160 Met ArgLys Val Glu Ser Leu Asn Leu Glu Lys Ala Ala Leu Thr Gln 165 170 175 GlnPro Gln Asn Ala Ala Thr Met Leu Thr Gln Glu Lys Lys Met Ala 180 185 190Leu Cys Glu Ile Cys Gly Ser Phe Leu Val Ala Asn Asp Ala Val Glu 195 200205 Arg Thr Gln Ser His Ile Thr Gly Lys Gln His Ile Gly Tyr Gly Met 210215 220 Val Arg Asp Tyr Leu Ala Glu Tyr Lys Glu Ala Lys Glu Lys Ala Arg225 230 235 240 Glu Glu Glu Arg Leu Ala Arg Glu Lys Glu Ala Glu Glu ArgArg Lys 245 250 255 Gln Arg Glu Lys Glu Asn Glu Ser Lys Asn Arg Arg SerIle Ser Ser 260 265 270 Glu Arg Asp Arg His Arg Asp Arg Asp Tyr Gly ArgAsp Arg Glu Arg 275 280 285 Ser Arg Glu Trp Asn Asn Arg Gly Asn Arg AspGlu Gly Arg Gly Met 290 295 300 Asp Arg Arg Arg Gln Tyr Asp Arg Asn GlyArg Asp Gly Gly Arg Asn 305 310 315 320 Thr Tyr His Gly Arg Glu Arg GluArg Ser Arg Ser Arg Ser Pro Val 325 330 335 Arg His Gly His Arg Arg 3409 650 DNA Beta vulgaris 9 acgaacaaca aaaatggcgg cagcagatgt tgaagcagtagacttcgaac ctgaagaaga 60 tgatctcatg gacgaagatg gcggtgcggc tgaagctgacggctctcctc gagctcctca 120 ccctaagatt aaatcagcca ttactggcgc cggagctccatcttctggcg gcttcggagc 180 taagaaaact aaaggtcgcg gcttccgtga agacgccgatgctgagcgta acagccgtat 240 gactgctcgt gaatttgatt ctcttgactc cgatggtggacctggtcctg ctcgatcaat 300 tgagggctgg attatacttg tcacgggagt gcatgaagaggctcaagaag aggatctcct 360 taatgtcttt ggagagtttg gccagcttaa gaatttgcatttgaatctgg atcgtcgtac 420 tgggtttgtc aagggttatg cattgatcga gtatgagaagtttgaagaag cacaagctgc 480 aataaaggag atgaatggtg ccaaaatgct tgagcagccgataaatgttg attgggcatt 540 ctgcaatggt ccttacagga ggaggggcaa ccgaagaagatccccacgtg gtcaccgatc 600 aaggagtcct agaagaagat attaaatctg tttgctgcatgtggaagttg 650 10 203 PRT Beta vulgaris 10 Met Ala Ala Ala Asp Val GluAla Val Asp Phe Glu Pro Glu Glu Asp 1 5 10 15 Asp Leu Met Asp Glu AspGly Gly Ala Ala Glu Ala Asp Gly Ser Pro 20 25 30 Arg Ala Pro His Pro LysIle Lys Ser Ala Ile Thr Gly Ala Gly Ala 35 40 45 Pro Ser Ser Gly Gly PheGly Ala Lys Lys Thr Lys Gly Arg Gly Phe 50 55 60 Arg Glu Asp Ala Asp AlaGlu Arg Asn Ser Arg Met Thr Ala Arg Glu 65 70 75 80 Phe Asp Ser Leu AspSer Asp Gly Gly Pro Gly Pro Ala Arg Ser Ile 85 90 95 Glu Gly Trp Ile IleLeu Val Thr Gly Val His Glu Glu Ala Gln Glu 100 105 110 Glu Asp Leu LeuAsn Val Phe Gly Glu Phe Gly Gln Leu Lys Asn Leu 115 120 125 His Leu AsnLeu Asp Arg Arg Thr Gly Phe Val Lys Gly Tyr Ala Leu 130 135 140 Ile GluTyr Glu Lys Phe Glu Glu Ala Gln Ala Ala Ile Lys Glu Met 145 150 155 160Asn Gly Ala Lys Met Leu Glu Gln Pro Ile Asn Val Asp Trp Ala Phe 165 170175 Cys Asn Gly Pro Tyr Arg Arg Arg Gly Asn Arg Arg Arg Ser Pro Arg 180185 190 Gly His Arg Ser Arg Ser Pro Arg Arg Arg Tyr 195 200 11 1464 DNABeta vulgaris 11 ttttgtagca tttgattttt gctgaaaaac ccaattcata ttttgaagaaatgacaacaa 60 tgaacccttt tgatttgttg ggtgacaatg ataacgatga cccatctcagcttttagagt 120 ctgcaactgc tcagttgcag aaaattgctg ttaaaaaaac cccaactcaggttgctcaac 180 aacctcagca acagaaagct gcaaagttac ccaccaaacc tcttcctccaactcaagctg 240 tccgggaggc aaagaatgat tcccagcgcg gaggggggcg tggaggaggtcgcggtagtg 300 gccgtgggcg tggtggatac aatagggact actcaaacaa tgaaaatgcttttaacagca 360 ctggagtaac tggcagtcaa ggggatgatg gggaaaggga aaggcgaccttatgcgggac 420 ctcgtggccc ttatcgtggt ggtcgccgag atgggttcaa caatgaggagggaagagacg 480 gggaacgccc gcgtagaacc tatgagcgac gaagtgggac tgggcgtggaagtgagatca 540 aacgtgaggg agcaggacgt ggaaactggg gtgctgaatc agatgaagttgcaccggtta 600 ctgaggaagc tggagaacaa aatgagaaga agttgaaccc tgagaatcttccagctgtag 660 aagatgctgc tgatggcatc aaggagggcc agccagatga gactgaagaaaaggaaccag 720 aggaaaagga gatgacactt gaagagtatg agaagttgct ggaagagaagaggaaggctt 780 tatcagcact caaggctgag gaacgcaagg tggaggttga caaagatttcgagtccatgc 840 aacagcttat aaacaaaaaa aaggatgaag actcagtttt catcaaattgggttctgaca 900 aggataagaa gaaggaagca gctgaaaagg agaaagtgaa gaagtctgtcagcattaatg 960 aatttctgaa gcctgctgaa ggggatagat atggtggtcg tggcaggggacgtggtcgtg 1020 gcccaagagg tggtggatat ggtggaggta ataggatgtt tagtacgtctgctccagcaa 1080 tcgaagatcc aggggagttc ccaaccctag gtggcaagtg aggccacatctttgaacttt 1140 ggtctctatt tggggtttta cttgaccccc tctgatttta agtcatttgagtgacaggaa 1200 tggacttcca gctgtgggtt tcctgtacca aatccacttt taagaaaatttttatgcttt 1260 ttaaatttgt atatttattc tgttaaaaaa aaaaaaaaaa actcgtgccgaattcgatat 1320 caagcttatc gataccgtcg acctcgaggg ggggcccggt accaagatggcctttggtgg 1380 gttgaagaag gaaaaagaca gaaacgactt aattacctac ttgaaaaaagcctgtgagta 1440 aacaggcccc ttttcctttg tcga 1464 12 356 PRT Beta vulgaris12 Met Thr Thr Met Asn Pro Phe Asp Leu Leu Gly Asp Asn Asp Asn Asp 1 510 15 Asp Pro Ser Gln Leu Leu Glu Ser Ala Thr Ala Gln Leu Gln Lys Ile 2025 30 Ala Val Lys Lys Thr Pro Thr Gln Val Ala Gln Gln Pro Gln Gln Gln 3540 45 Lys Ala Ala Lys Leu Pro Thr Lys Pro Leu Pro Pro Thr Gln Ala Val 5055 60 Arg Glu Ala Lys Asn Asp Ser Gln Arg Gly Gly Gly Arg Gly Gly Gly 6570 75 80 Arg Gly Ser Gly Arg Gly Arg Gly Gly Tyr Asn Arg Asp Tyr Ser Asn85 90 95 Asn Glu Asn Ala Phe Asn Ser Thr Gly Val Thr Gly Ser Gln Gly Asp100 105 110 Asp Gly Glu Arg Glu Arg Arg Pro Tyr Ala Gly Pro Arg Gly ProTyr 115 120 125 Arg Gly Gly Arg Arg Asp Gly Phe Asn Asn Glu Glu Gly ArgAsp Gly 130 135 140 Glu Arg Pro Arg Arg Thr Tyr Glu Arg Arg Ser Gly ThrGly Arg Gly 145 150 155 160 Ser Glu Ile Lys Arg Glu Gly Ala Gly Arg GlyAsn Trp Gly Ala Glu 165 170 175 Ser Asp Glu Val Ala Pro Val Thr Glu GluAla Gly Glu Gln Asn Glu 180 185 190 Lys Lys Leu Asn Pro Glu Asn Leu ProAla Val Glu Asp Ala Ala Asp 195 200 205 Gly Ile Lys Glu Gly Gln Pro AspGlu Thr Glu Glu Lys Glu Pro Glu 210 215 220 Glu Lys Glu Met Thr Leu GluGlu Tyr Glu Lys Leu Leu Glu Glu Lys 225 230 235 240 Arg Lys Ala Leu SerAla Leu Lys Ala Glu Glu Arg Lys Val Glu Val 245 250 255 Asp Lys Asp PheGlu Ser Met Gln Gln Leu Ile Asn Lys Lys Lys Asp 260 265 270 Glu Asp SerVal Phe Ile Lys Leu Gly Ser Asp Lys Asp Lys Lys Lys 275 280 285 Glu AlaAla Glu Lys Glu Lys Val Lys Lys Ser Val Ser Ile Asn Glu 290 295 300 PheLeu Lys Pro Ala Glu Gly Asp Arg Tyr Gly Gly Arg Gly Arg Gly 305 310 315320 Arg Gly Arg Gly Pro Arg Gly Gly Gly Tyr Gly Gly Gly Asn Arg Met 325330 335 Phe Ser Thr Ser Ala Pro Ala Ile Glu Asp Pro Gly Glu Phe Pro Thr340 345 350 Leu Gly Gly Lys 355 13 1310 DNA Beta vulgaris 13 ccgacaagttagggtttttc caagggtttt ctgaaaaaca gcgaataaca atggcaacca 60 ctaaccctttcgacttgctc gacgacgatg ctgaggaccc agccctcctt attgctgcgc 120 aggagcagaaggtttccgcc gtcgttgccg gagataagaa aactccggca gtcgctgcta 180 agcctgctaaactccctact aagcctcttc ctccttctca agctgtgaga gaggcaagga 240 atgatggtggtcgtggtgga ggtggccgcg ggggccgtgg ttatgggcgg ggacgtggtc 300 caggtggacctaatagagat tcaacaaata atgatgaaat atatcccaac gagaatgggg 360 gttctatgggatatagggag gacagagata agccatctga aagacgtgga ggatatggcg 420 gtcctcgtggtggttatcgt ggaggacgac gtggaggtta tgataatgga gaagctgctg 480 aaggagaacgtcctaggagg atgtatgaac gccgtagtgg cactggacga ggaggtgaga 540 ttaaacgtgagggttctggt cgtggaaact ggggatctcc tactgatgag atagctccgg 600 agactgaagaacctgttgtg gaaaatgaag cagctgttgc agctgataag ccagcaggag 660 agggagaaaatgttgatgct gaaaaggaga gtcaagagaa ggaagttgta gaagcagagc 720 ctgaagaaaaggaaatgact cttgaggagt atgagaaggt attggaggag aagaggaagg 780 ccttgctatcattgaaaggg gaggaaagaa aggtggattt ggacaaggag tttgaatcta 840 tgcagctggtttcaaagaag aagaatgatg atgaggtttt cataaagctg ggttctgata 900 aggacaagagaaaggaggct gcagaaagag aagaaaggtc caagaagtct gtgagcatca 960 atgaatttcttaagcctgcc gagggtgacg gataccacag gcgtggaaga ggaagaggcc 1020 gtggtggtaggggaggctat ggtggaggat acggcatgaa caatgcatct gctccttcta 1080 ttgaggatcccaatcaattc ccatctttgg gtgcgaactg agtttttgtc cgttgttgtc 1140 ttagttatttttgggtcttt cttatatttt gagacttatt tatgatgttc aggagcctca 1200 tcaattacaaaaaaagatat ttgacaggaa taatgtgttt ttcctgtgtt aagagtgtaa 1260 atcttagatgtttcatcttt caaaaaaaaa aaaaaaaact cgaggggggg 1310 14 356 PRT Betavulgaris 14 Met Ala Thr Thr Asn Pro Phe Asp Leu Leu Asp Asp Asp Ala GluAsp 1 5 10 15 Pro Ala Leu Leu Ile Ala Ala Gln Glu Gln Lys Val Ser AlaVal Val 20 25 30 Ala Gly Asp Lys Lys Thr Pro Ala Val Ala Ala Lys Pro AlaLys Leu 35 40 45 Pro Thr Lys Pro Leu Pro Pro Ser Gln Ala Val Arg Glu AlaArg Asn 50 55 60 Asp Gly Gly Arg Gly Gly Gly Gly Arg Gly Gly Arg Gly TyrGly Arg 65 70 75 80 Gly Arg Gly Pro Gly Gly Pro Asn Arg Asp Ser Thr AsnAsn Asp Glu 85 90 95 Ile Tyr Pro Asn Glu Asn Gly Gly Ser Met Gly Tyr ArgGlu Asp Arg 100 105 110 Asp Lys Pro Ser Glu Arg Arg Gly Gly Tyr Gly GlyPro Arg Gly Gly 115 120 125 Tyr Arg Gly Gly Arg Arg Gly Gly Tyr Asp AsnGly Glu Ala Ala Glu 130 135 140 Gly Glu Arg Pro Arg Arg Met Tyr Glu ArgArg Ser Gly Thr Gly Arg 145 150 155 160 Gly Gly Glu Ile Lys Arg Glu GlySer Gly Arg Gly Asn Trp Gly Ser 165 170 175 Pro Thr Asp Glu Ile Ala ProGlu Thr Glu Glu Pro Val Val Glu Asn 180 185 190 Glu Ala Ala Val Ala AlaAsp Lys Pro Ala Gly Glu Gly Glu Asn Val 195 200 205 Asp Ala Glu Lys GluSer Gln Glu Lys Glu Val Val Glu Ala Glu Pro 210 215 220 Glu Glu Lys GluMet Thr Leu Glu Glu Tyr Glu Lys Val Leu Glu Glu 225 230 235 240 Lys ArgLys Ala Leu Leu Ser Leu Lys Gly Glu Glu Arg Lys Val Asp 245 250 255 LeuAsp Lys Glu Phe Glu Ser Met Gln Leu Val Ser Lys Lys Lys Asn 260 265 270Asp Asp Glu Val Phe Ile Lys Leu Gly Ser Asp Lys Asp Lys Arg Lys 275 280285 Glu Ala Ala Glu Arg Glu Glu Arg Ser Lys Lys Ser Val Ser Ile Asn 290295 300 Glu Phe Leu Lys Pro Ala Glu Gly Asp Gly Tyr His Arg Arg Gly Arg305 310 315 320 Gly Arg Gly Arg Gly Gly Arg Gly Gly Tyr Gly Gly Gly TyrGly Met 325 330 335 Asn Asn Ala Ser Ala Pro Ser Ile Glu Asp Pro Asn GlnPhe Pro Ser 340 345 350 Leu Gly Ala Asn 355 15 1155 DNA Beta vulgaris 15gatggacgag aattatattc gtacttgttt cgctcaatcc ggcgagcttg ttaatgttaa 60aatcatccgt aataagcaaa ccatgcagtc agagtgctat ggatttattg agttttccac 120ccatgctgct gctgaaagga ttttgcagac ttacaataac accttgatgc caaatgttga 180gcaaaactac agactgaatt gggctttcta tggatctggt gagaagcgtg gagaggatgc 240ttctgattat acaatttttg ttggggattt agctccagat gttactgatt acacattgca 300agagacattt agagttcgct atccatctgt aaaaggtgct aaggttgtga tagatagact 360gacaagtaga tcaaagggtt atggatttgt tcgtttcgga gatgaaagtg aacaagcacg 420tgccatgtca gagatgaatg gaatgatgtg cttaggccgt gcaatgcgta ttggagcagc 480tgcaaacaag aaaagtgttg gcggaacagc ttcatatcag aataatcagg gaactccaaa 540tgacagtgat ccgagtaaca ctactatatt tgttggcaat ttggattcta atgtgactga 600tgaacatttg agacaaacat ttagccctta cggagaattg gtccatgtaa aaattcctgc 660gggcaaacag tgcgggtttg ttcaatttac taacagaagt agtgctgagg aagcattgag 720ggtattgaac ggaatgcaat taggcggacg aaatgttaga ctttcgtggg gccgtagtcc 780taacaacaga cagtctcaac ctgaccagaa ccagtggaac aatgctgctt attatggtta 840tcctcaagga tacgactctt atggatatgt atctgctcct caagacccaa acatgtacta 900tggtggctac cctggttatg gtggttacgc gatgcctcag caggctcaga tgccattgca 960acaacagtga tctaccttat gccaagcagg agaggtcggt tgccagggag ctgtcattgt 1020acttggaggc tgagcttctg gagttggatg attcctccca gagatggcag aatgtagtat 1080aacttggtca ttgtgctggt cgaattttat ttactgtctt gggtttttgc tctgtgctgc 1140ttttttgtag cttgc 1155 16 322 PRT Beta vulgaris 16 Met Asp Glu Asn TyrIle Arg Thr Cys Phe Ala Gln Ser Gly Glu Leu 1 5 10 15 Val Asn Val LysIle Ile Arg Asn Lys Gln Thr Met Gln Ser Glu Cys 20 25 30 Tyr Gly Phe IleGlu Phe Ser Thr His Ala Ala Ala Glu Arg Ile Leu 35 40 45 Gln Thr Tyr AsnAsn Thr Leu Met Pro Asn Val Glu Gln Asn Tyr Arg 50 55 60 Leu Asn Trp AlaPhe Tyr Gly Ser Gly Glu Lys Arg Gly Glu Asp Ala 65 70 75 80 Ser Asp TyrThr Ile Phe Val Gly Asp Leu Ala Pro Asp Val Thr Asp 85 90 95 Tyr Thr LeuGln Glu Thr Phe Arg Val Arg Tyr Pro Ser Val Lys Gly 100 105 110 Ala LysVal Val Ile Asp Arg Leu Thr Ser Arg Ser Lys Gly Tyr Gly 115 120 125 PheVal Arg Phe Gly Asp Glu Ser Glu Gln Ala Arg Ala Met Ser Glu 130 135 140Met Asn Gly Met Met Cys Leu Gly Arg Ala Met Arg Ile Gly Ala Ala 145 150155 160 Ala Asn Lys Lys Ser Val Gly Gly Thr Ala Ser Tyr Gln Asn Asn Gln165 170 175 Gly Thr Pro Asn Asp Ser Asp Pro Ser Asn Thr Thr Ile Phe ValGly 180 185 190 Asn Leu Asp Ser Asn Val Thr Asp Glu His Leu Arg Gln ThrPhe Ser 195 200 205 Pro Tyr Gly Glu Leu Val His Val Lys Ile Pro Ala GlyLys Gln Cys 210 215 220 Gly Phe Val Gln Phe Thr Asn Arg Ser Ser Ala GluGlu Ala Leu Arg 225 230 235 240 Val Leu Asn Gly Met Gln Leu Gly Gly ArgAsn Val Arg Leu Ser Trp 245 250 255 Gly Arg Ser Pro Asn Asn Arg Gln SerGln Pro Asp Gln Asn Gln Trp 260 265 270 Asn Asn Ala Ala Tyr Tyr Gly TyrPro Gln Gly Tyr Asp Ser Tyr Gly 275 280 285 Tyr Val Ser Ala Pro Gln AspPro Asn Met Tyr Tyr Gly Gly Tyr Pro 290 295 300 Gly Tyr Gly Gly Tyr AlaMet Pro Gln Gln Ala Gln Met Pro Leu Gln 305 310 315 320 Gln Gln 17 1200DNA Beta vulgaris 17 aaaaacctct ttttctctct cctaaatcac aacaatggcgatactctcag attacgagga 60 agaagaacac caaccacaac cagaaaagaa gcaaccttcaaagaaatttt cagcaacttt 120 cgatccttcg aatccgctag ggtttcttca atctactctcgaattcgtct caaaagagtc 180 cgattttttc gctaaggaat catctgcgaa agatgttgtttctctggttc agaaagtgaa 240 ggagaagtac attgaagaag tagagaataa gaagaagaagcttctagatg aatctgccgc 300 tgccgccgcc gccgccgctg ctgctgctgc gtcgtcgtcttcatctgatt tggagaagaa 360 ggttgatgat aatgagagtg cggaagagac agagaaatctaagtacaaag ctccaaacag 420 tgggaatggt caagatctcg agaactactc atggatacagtccttgcaag aagttactgt 480 taatgttcct gttccacctg gaacaaagtc taggtttatcgattgtcaga taaagaagaa 540 tcatctgaaa gttggcctca agggtcagcc tcccatcatcgatggtgaac tgttcaagcc 600 tgttaagcca gatgattgtt tttggagttt ggaggatcaaaagtcaatct ctatgctgct 660 aacaaagcat gatcaaatgg agtggtggag aagtctggtcaaaggtgaac ctgaaatcga 720 cactcagaag gttgaacctg agagcagtaa gctgtctgacttggaccctg aaacaaggtc 780 aactgttgag aagatgatgt ttgaccaaag gcaaaaatccatgggcttgc ccacaagtga 840 tgatatgcag aagcaagaca tgctgaagaa gttcatgtccgagcatccgg aaatggactt 900 ttctaacgcg aagtttaact agatatcgat gtcggtgatggactatgatt ttttgggtgg 960 caaattctcg aaacaggaac tgaagaaagc ttttgttatgtctaatactg agcttgttca 1020 tagtagttac agtctctagg gtagatgtct catgaagaggggaacattgc tttttgttta 1080 actcttattt atatgcaagt gatattcggt ttgctaagcagtacattcgt gcatcctgcg 1140 cttgattcgg gtcctgttca atcatatatg taatgttatagctgcaaaaa aaaaaaaaaa 1200 18 295 PRT Beta vulgaris 18 Met Ala Ile LeuSer Asp Tyr Glu Glu Glu Glu His Gln Pro Gln Pro 1 5 10 15 Glu Lys LysGln Pro Ser Lys Lys Phe Ser Ala Thr Phe Asp Pro Ser 20 25 30 Asn Pro LeuGly Phe Leu Gln Ser Thr Leu Glu Phe Val Ser Lys Glu 35 40 45 Ser Asp PhePhe Ala Lys Glu Ser Ser Ala Lys Asp Val Val Ser Leu 50 55 60 Val Gln LysVal Lys Glu Lys Tyr Ile Glu Glu Val Glu Asn Lys Lys 65 70 75 80 Lys LysLeu Leu Asp Glu Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 85 90 95 Ala AlaAla Ser Ser Ser Ser Ser Asp Leu Glu Lys Lys Val Asp Asp 100 105 110 AsnGlu Ser Ala Glu Glu Thr Glu Lys Ser Lys Tyr Lys Ala Pro Asn 115 120 125Ser Gly Asn Gly Gln Asp Leu Glu Asn Tyr Ser Trp Ile Gln Ser Leu 130 135140 Gln Glu Val Thr Val Asn Val Pro Val Pro Pro Gly Thr Lys Ser Arg 145150 155 160 Phe Ile Asp Cys Gln Ile Lys Lys Asn His Leu Lys Val Gly LeuLys 165 170 175 Gly Gln Pro Pro Ile Ile Asp Gly Glu Leu Phe Lys Pro ValLys Pro 180 185 190 Asp Asp Cys Phe Trp Ser Leu Glu Asp Gln Lys Ser IleSer Met Leu 195 200 205 Leu Thr Lys His Asp Gln Met Glu Trp Trp Arg SerLeu Val Lys Gly 210 215 220 Glu Pro Glu Ile Asp Thr Gln Lys Val Glu ProGlu Ser Ser Lys Leu 225 230 235 240 Ser Asp Leu Asp Pro Glu Thr Arg SerThr Val Glu Lys Met Met Phe 245 250 255 Asp Gln Arg Gln Lys Ser Met GlyLeu Pro Thr Ser Asp Asp Met Gln 260 265 270 Lys Gln Asp Met Leu Lys LysPhe Met Ser Glu His Pro Glu Met Asp 275 280 285 Phe Ser Asn Ala Lys PheAsn 290 295 19 1050 DNA Mus musculus 19 gaaaagagag gagagagaaa accaaaatcaacaaaaatgg cggaacatct agcatcgata 60 ttcgggacag agaaagacag agtgaactgtccattctact tcaagatcgg agcttgtaga 120 catggagatc gttgctcaag gcttcatactaagcctagta ttagccctac tttgttgctt 180 gctaatatgt atcaacgccc tgatatgattactcctggtg ttgatcctca aggacagcct 240 cttgatcctc gcaaaattca acaacattttgaggattttt atgaggattt atttgaggaa 300 ctaagcaagt atggggagat tgaaagtctcaacatctgtg acaatttggc tgaccacatg 360 gttgggaatg tttatgtgca gttcagagaggaagaacatg ctggcgaggc actacgaaac 420 ttgagtggaa gattttatgc cggtcgtccaatcattgttg atttttctcc tgtaacggac 480 ttcagagaag caacctgcag acagtatgaggaaaatgtgt gcaatcgtgg aggttactgc 540 aactttatgc atttgaaaaa aattagcagggagcttaggc gacagttgtt tggaaggtac 600 agaaggaggc atagccgtag tagaagtcgcagtcctcaag cacatcgggg gcatggagat 660 cgtccacatg gtggccgtgg ttatggtagaagagatgatg atagaaatca gcggtaccat 720 gacaagggaa gaaggcctag aagccgtagccctgggcata gaggacgaag cagaagccct 780 cccggcagga gggataggag tccagtgagggagaatagtg aggagagaag agcaaagatt 840 gcacaatgga acagggaaaa ggaacaggcagacactggta ataacgatgt taatcatgat 900 gtcactgaca accatgcaaa tggatttcaggacaatgggg aggattacta tgaccatcct 960 cagcagtaac tggatgaagt gcacaagcaggctttattca ctacttctgg tttgctgtta 1020 tcagagtctg ctcgtttgca ggatttttcg1050 20 310 PRT Mus musculus 20 Met Ala Glu His Leu Ala Ser Ile Phe GlyThr Glu Lys Asp Arg Val 1 5 10 15 Asn Cys Pro Phe Tyr Phe Lys Ile GlyAla Cys Arg His Gly Asp Arg 20 25 30 Cys Ser Arg Leu His Thr Lys Pro SerIle Ser Pro Thr Leu Leu Leu 35 40 45 Ala Asn Met Tyr Gln Arg Pro Asp MetIle Thr Pro Gly Val Asp Pro 50 55 60 Gln Gly Gln Pro Leu Asp Pro Arg LysIle Gln Gln His Phe Glu Asp 65 70 75 80 Phe Tyr Glu Asp Leu Phe Glu GluLeu Ser Lys Tyr Gly Glu Ile Glu 85 90 95 Ser Leu Asn Ile Cys Asp Asn LeuAla Asp His Met Val Gly Asn Val 100 105 110 Tyr Val Gln Phe Arg Glu GluGlu His Ala Gly Glu Ala Leu Arg Asn 115 120 125 Leu Ser Gly Arg Phe TyrAla Gly Arg Pro Ile Ile Val Asp Phe Ser 130 135 140 Pro Val Thr Asp PheArg Glu Ala Thr Cys Arg Gln Tyr Glu Glu Asn 145 150 155 160 Val Cys AsnArg Gly Gly Tyr Cys Asn Phe Met His Leu Lys Lys Ile 165 170 175 Ser ArgGlu Leu Arg Arg Gln Leu Phe Gly Arg Tyr Arg Arg Arg His 180 185 190 SerArg Ser Arg Ser Arg Ser Pro Gln Ala His Arg Gly His Gly Asp 195 200 205Arg Pro His Gly Gly Arg Gly Tyr Gly Arg Arg Asp Asp Asp Arg Asn 210 215220 Gln Arg Tyr His Asp Lys Gly Arg Arg Pro Arg Ser Arg Ser Pro Gly 225230 235 240 His Arg Gly Arg Ser Arg Ser Pro Pro Gly Arg Arg Asp Arg SerPro 245 250 255 Val Arg Glu Asn Ser Glu Glu Arg Arg Ala Lys Ile Ala GlnTrp Asn 260 265 270 Arg Glu Lys Glu Gln Ala Asp Thr Gly Asn Asn Asp ValAsn His Asp 275 280 285 Val Thr Asp Asn His Ala Asn Gly Phe Gln Asp AsnGly Glu Asp Tyr 290 295 300 Tyr Asp His Pro Gln Gln 305 310 21 117 PRTArabidopsis thaliana 21 Met Val Thr Thr Pro His Glu Lys Ala Thr Asp SerLys Lys Ser Gly 1 5 10 15 Thr Glu Ser Asn Ser Gln Pro Ile Val Gly AspSer Ser Tyr Glu Arg 20 25 30 Ser Lys Val Gly Asp Arg Glu Arg Glu Ser AspArg Glu Lys Glu Arg 35 40 45 Gly Arg Glu Arg Asp Arg Gly Arg Ser His ArgGly Arg Asp Ser Asp 50 55 60 Arg Asp Ser Asp Arg Glu Arg Asp Lys Leu LysAsp Arg Ser His His 65 70 75 80 Arg Ser Arg Asp Arg Leu Lys Asp Ser GlyGly His Ser Asp Lys Ser 85 90 95 Arg His His Ser Ser Arg Asp Arg Asp TyrArg Asp Ser Ser Lys Asp 100 105 110 Arg Arg Arg His His 115 22 55 PRTArabidopsis thaliana 22 Met Ile Tyr Thr Ala Ile Asp Asn Phe Tyr Leu ThrAsp Glu Gln Leu 1 5 10 15 Lys Ala Ser Pro Ser Arg Lys Asp Gly Ile AspGlu Thr Thr Glu Ile 20 25 30 Ser Leu Arg Ile Tyr Gly Cys Asp Leu Ile GlnGlu Gly Gly Ile Leu 35 40 45 Leu Lys Leu Pro Gln Ala Val 50 55 23 17 DNAArtificial Sequence Description of Artificial SequenceProbe or Primer 23catcaaatct ttcaggg 17 24 18 DNA Artificial Sequence Description ofArtificial SequenceProbe or Primer 24 ctttatttta ctgtacag 18

1. A method to enhance stress tolerance in cells and organismscomprising the manipulation of the process of processing messenger RNAprecursors.
 2. A method to enhance salt toxicity in cells and organismscomprising the manipulation of the process of processing messenger RNAprecursors.
 3. A method according to any of claims 1 or 2 wherein saidmanipulation comprises the genetic or biochemical manipulation of amolecule involved in or interfering with the process of processingmessenger RNA precursors.
 4. A method according to claim 1 or 3 whereinsaid stress tolerance comprises tolerance against osmotic, drought, coldor freezing stress.
 5. A method according to any of claims 1 to 4,comprising the genetic or biochemical manipulation of a proteinpossessing a domain with a high content in Arg-Ser, Arg-Glu and Arg-Aspdipeptides (RS domain).
 6. A method according to any of claims 1 to 4,comprising the genetic or biochemical manipulation of a protein of anRNA binding protein.
 7. A method according to any of claims 1 to 4,comprising the genetic or biochemical manipulation of a component of theU1-snRNP or the U2-snRNP complex.
 8. A method according to any of claims1 to 4, comprising the genetic or biochemical manipulation of atranscription factor.
 9. A method according to any of claims 1 to 4,comprising the genetic or biochemical manipulation of a nuclear movementprotein.
 10. Use of an isolated nucleic acid comprising a nucleic acidsequence as represented in SEQ ID NO 1 with an amino acid sequence asset forth in SEQ ID NO 3, or a nucleic acid as represented in SEQ ID NO2 with an amino acid sequence as set forth in SEQ ID NO 4 or SEQ ID NO21 or an amino acid sequence comprising SEQ ID NO 22, for any of themethods according to claims 1 to
 5. 11. Use of an isolated nucleic acidcomprising a nucleic acid sequence as represented in SEQ ID NO 5 with anamino acid sequence as set forth in SEQ ID NO 6 for any of the methodsof claims 1 to 4 or
 7. 12. Use of an isolated nucleic acid comprising anucleic acid sequence as represented in any of SEQ ID NOs 7, 9, 11, 13,15, 17 or 19, with an amino acid sequence as set forth in any of SEQ IDNO 8, 10, 12, 14, 16, 18 or 20 for any of the methods of claims 1 to 4.13. An isolated nucleic acid encoding a protein or an immunologicallyactive and/or functional fragment of such a protein selected from thegroup consisting of: a) a nucleic acid comprising a DNA sequence asgiven in any of SEQ ID NOs 1, 2, 5, 7, 9, 11, 13, 15, 17 or 19 or thecomplement thereof, b) Nucleic acid comprising the RNA sequencecorresponding to any of SEQ ID NOs 1, 2, 5, 7, 9, 11, 13, 15, 17 or 19as in (a) or the complement thereof, c) Nucleic acid specificallyhybridizing to the nucleotide sequence ad defined in (a) or (b), d)nucleic acid encoding a protein with an amino acid sequence which is atleast 95% identical to the polypeptide represented in any of SEQ ID NOs3, 4, 6, 8, 10, 12, 14, 16, 18, 20or 21, e) nucleic acid encoding aprotein comprising the amino acid sequence as given in any of SEQ IDNOs, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 21 or 22, f) nucleic acid whichis degenerated as a result of the genetic code to a nucleotide sequenceof a nucleic acid as given in any of SEQ ID NOs 1, 2, 5, 7, 9, 11, 13,15, 17 or 19 or as defined (a) to (e), g) nucleic acid which isdiverging due to the differences in codon usage between the organisms toa nucleotide sequnce encoding a protein as given in any of SEQ ID NOs 3,4, 6, 8, 10, 12, 14, 16, 18, 20 or 21 or as defined in (a) to (e), h)nucleic acid which is diverging due to the differences in allelesencoding a protein as given in any of SEQ ID NOs, 3, 4, 6, 8, 10, 12,14, 16, 18, 20 or 21 or as defined in (a) to (e), i) nucleic acidencoding an immunologically active and/or functional fragment of aprotein encoded by a DNA sequence as given in any of SEQ ID NOs 1, 2, 5,7, 9, 11, 13, 15, 17 or 19 or as defined (a) to (e), j) nucleic acidencoding a protein as defined in SEQ ID Nos, 3, 4, 6, 8, 10, 12, 14, 16,18, 20 or 21 or as defined in (a) to (i) characterized in that saidsequence is DNA, cDNA, genomic DNA or synthetic DNA.
 14. A nucleic acidmolecule of at least 15 nucleotides in length specifically hybridizingwith a nucleic acid of claim
 13. 15. A nucleic acid molecule of at least15 nucleotides in length specifically amplifying a nucleic acid of claim13.
 16. A vector comprising a nucleic acid according to claim
 13. 17. Avector according to claim 16 which Is an expression vector in whereinthe nucleic acid is operably linked to one or more control sequencesallowing the expression of said sequence in prokaryotic and/oreukaryotic host cells.
 18. A host cell containing a nucleic acidaccording to claim 13 or a vector according to claim 16 or
 17. 19. Ahost cell according to claim 17 chosen from a bacterial, insect, fungal,yeast, plant or animal cell.
 20. Use of a natural or synthetic nucleicacid encoding a protein containing an “RS domain” as defined in claim 5in any of the methods of claims 1 to 4 .
 21. Use of a natural orsynthetic nucleic acid encoding a protein involved in the process ofprocessing messenger RNA precursors in eukaryotic cells in any of themethods of claims 1 to
 9. 22. Use of a nucleic acid with at least 50%identity to at least one of the sequences identified in any of claims 10to 13 in a method of claim 1, wherein said nucleic acid is derived froman eukaryotic cell.
 23. Anti-sense molecule corresponding to at leastone of the nucleic acids defined in claim
 12. 24. Use of an anti-sensemolecule of claim 23 in any of the methods of claims 1 to
 4. 25. Apolypeptide encodable by at least one of the nucleic acids defined inclaim 13, or a homologue thereof or a derivative thereof, or animmunologically active and/or functional fragment thereof.
 26. Thepolypeptide of claim 25 comprising an amino acid sequence as given inany of SEQ ID NOs 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 21 or 22 or ahomologue thereof or a derivative thereof, or an immunologically activeand/or functional fragment thereof.
 27. A method of producing apolypeptide according to claim 25 or 26 comprising culturing a host cellof claim 18 or 19 under the conditions allowing the expression of thepolypeptide and recovering the produced polypeptide from the culture.28. A method for the production of transgenic plants, plant cells orplant tissues comprising the introduction of a nucleic acid moleculeaccording to claim 13 in an expressible format or a vector according toclaim 16 or 17 in said plant, plant cell or plant tissue.
 29. A methodfor the production of altered plants, plant cells or plant tissuescomprising the introduction of a polypeptide of claim 25 or 26 directlyinto a cell, a tissue or an organ of said plant.
 30. A method foreffecting the expression of a polypeptide of claim 25 or 26 comprisingthe introduction of a nucleic acid of claim 13 operably linked to one ormore control sequences or a vector according to claim 16 or 17 stablyinto the genome of a plant cell.
 31. The method of claim 28 or 30further comprising regenerating a plant from said plant cell.
 32. Thetransgenic plant cell obtainable by a method of claim 31 wherein saidnucleic acid is stably integrated into the genome of said plant cell.33. Transgenic plants tolerant to salt stress as a result of theexpression of at least one of the nucleic acids of claim 13 or at leastone of the polypeptides of claim 25 or 26 or an anti-sense molecule ofclaim
 23. 34. Transgenic plants which as a result of the expression ofat least one of the nucleic acids of claim 13 or at least one of thepolypeptides of claim 25 or 26 or an anti-sense molecule of claim 23show an alteration of their phenotype.
 35. A harvestable part of a plantof claim 33 and
 34. 36. A harvestable part of a plant of claim 35 whichis selected from the group consisting of seeds, leaves, fruits, stemcultures, rhizomes, roots, tubers and bulbs.
 37. The progeny derivedfrom any of the plants or plant parts of any of claims 33 to
 36. 38. Amethod for enhancing stress tolerance in (a) plant(s) comprisingexpression of at least one of the nucleic acids of claim 13 or at leastone of the polypeptides of claim 25 or 26 or an anti-sense molecule ofclaim 23 in cells, tissues or parts of said plant(s).
 39. A method foraltering stress tolerance in (a) plant(s) comprising expression of atleast one of the nucleic acids of claim 13 or at least one of thepolypeptides of claim 25 or 26 or an anti-sense molecule of claim 23 incells, tissues or parts of said plant(s).
 40. A method of claim 38 or39, wherein said stress is osmotic stress.
 41. A method of claim 38 or39, wherein said stress is salt stress
 42. A method of claim 38 or 39,wherein said stress is drought stress
 43. A method of claim 38 or 39,wherein said stress is freezing stress
 44. A method of claim 38 or 39,wherein said stress is cold stress
 45. A method of any of claims 1 to 9,27 to 31 or 38 to 44 for increasing yield.
 46. Use of a nucleic acid ofclaim 13, a vector of claim 16 or 17, a polypeptide of claim 25 or 26for increasing yield.
 47. Use of a nucleic acid of claim. 13, a vectorof claim 16 or 17, a polypeptide of claim 25 or 26 for stimulating plantgrowth, which can be in any part of that plant, such as root, leave,seed.
 48. Use of a plant obtainable by any of the methods of claims 1 to9, 28 to 31 or 38 to 42 or the plant of claim 33 or 34 for culturing onsoil with a salt content of more than 1 mM salt ions.
 49. New strains ofyeast or other unicellular eukaryotes more tolerant to salt stress as aresult of the expression of any of the nucleic acids of claim 13, and/orproteins of claims 25 or
 26. 50. An in vitro cell culture systemcomprising animal, plant or host cells as defined in claims 18 or 19,tolerant to salt, obtained as a result of the expression of at least oneof the nucleic acids claim of 13, a vector of claim 16 or 17, apolypeptide of claim 25 or 26 or an antisense molecule of claim
 23. 51.A therapeutic application in humans derived from the method described inclaim
 1. 52. An antibody specifically recognizing a polypeptide of claim25 or 26 or a specific epitope thereof.
 53. Diagnostic compositioncomprising at least a nucleic acid of claims 13 to 15, a vector of claim16 or 17, a polypeptide of claim 25 or 26 or an antibody of claim 52.